Recombinant low virulence bovine herpesvirus-1 (BoHV-1) vaccine vectors

ABSTRACT

The present disclosure teaches generally in the field of vaccination and disease control in cattle and bovine animals. A recombinant bovine herpesvirus 1 (BoHV-1) vaccine vector is provided for efficient control of one or more bovine pathogens such as those associated with bovine respiratory disease complex, such as bovine viral diarrhea virus (BVDV), and which ameliorates disease conditions caused thereby. Protocols for the management of confined or herded bovine animals are also enabled herein.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 14/130,904, filed Feb. 3, 2014, which is U.S. national phaseapplication of PCT/AU2012/000804, which claims priority from AustralianProvisional Patent Application No. 2011902660, filed on 5 Jul. 2011,entitled “A vaccine,” the entire contents of which, are incorporatedherein by reference.

REFERENCE TO SEQUENCE LISTING

A Sequence Listing submitted as an ASCII text file via EFS-Web is herebyincorporated by reference in accordance with 35 U.S.C. § 1.52(e). Thename of the ASCII text file for the Sequence Listing is 23429554_1.TXT,the date of creation of the ASCII text file is May 31, 2016, and thesize of the ASCII text file is 2.98 KB.

FIELD

The present disclosure teaches generally in the field of vaccination anddisease control in bovine animals. A vaccine vector is provided forefficient control of one or more bovine pathogens such as thoseassociated with bovine respiratory disease complex and which amelioratesdisease conditions caused thereby. Protocols for the management ofconfined or herded bovine animals are also enabled herein.

BACKGROUND

Bibliographic details of the publications referred to by author in thisspecification are collected alphabetically at the end of thedescription.

Reference to any prior art in this specification is not, and should notbe taken as, an acknowledgment or any form of suggestion that this priorart forms part of the common general knowledge in any country.

Bovine Respiratory disease complex (BRDC) is the most significantinfectious disease of feedlot cattle in Australia. BRDC causes economicloss due to morbidity, mortality, loss of feed resources, medicationpurchases, increased time on feed and associated labor costs. BRDC has acomplicated etiology with at least four viral and three bacterialspecies along with environmental conditions predisposing an animal tothe illness.

The four viruses associated with BRDC are bovine herpesvirus 1 (BoHV-1),bovine viral diarrhea virus (BVDV or bovine pestivirus), bovineparainfluenza 3 virus and bovine respiratory syncytial virus.Serological surveys have shown that all of these viruses infect feedlotcattle in Australia. Three bacterial species, Pasteurella mutocida,Manhiemia haemolytica and Haemophilus somnus, have also been implicatedin BRDC.

In North America and in Europe, both live and killed vaccines have beenused to control diseases caused by BoHV-1. These vaccines are based ondifferent genotypes of BoHV-1 to that found in Australia. North Americanand European BoHV-1 strains are generally classified into the subgroup1.1 while Australian strains form the subgroup 1.2. The BoHV-1.1 virusescause a more severe clinical disease compared to the BoHV-1.2 viruses.The exact molecular mechanism for this difference in phenotype isunknown.

BoHV-1 is a virus of the family Herpesviridae that causes severaldiseases worldwide in cattle, including rhinotracheitis, vaginitis,balanoposthitis, abortion, conjunctivitis and enteritis. BoHV-1 is alsoa contributing factor in shipping fever. It is spread through sexualcontact, artificial insemination and aerosol transmission. Like otherherpesviruses, BoHV-1 causes a lifelong latent infection and shedding ofthe virus. The sciatic nerve and trigeminal nerve are the sites oflatency.

The respiratory disease caused by BoHV-1 is commonly known as infectiousbovine rhibotracheitis. Symptoms include fever, discharge from the nose,cough, difficulty in breathing and loss of appetite. Ulcers commonlyoccur in the mouth and nose. Mortality rates may reach 10 percent. Thegenital disease causes infectious pustular vulvovaginitis in cows andinfectious balanoposthitis in bulls. Symptoms include fever, depression,loss of appetite, painful urination, a swollen vulva with pustules anddischarge in cows and pain on sexual contact in bulls. In both cases,lesions usually resolve within two weeks. Abortion and stillbirths canoccur one to three months post infection. BoHV-1 also causes ageneralized disease in newborn calves, characterized by enteritis anddeath.

Similarly, BVDV is a disease of cattle which reduces productivity andincreases mortality. It is caused by a pestivirus from the familyFlaviviridae. Pestiviruses have the ability to establish persistentinfection during pregnancy. Persistent infection with pestiviruses oftengoes unnoticed. BVDV also frequently undergoes non-homologous RNArecombination leading to the appearance of genetically distinct virusesthat are lethal to the host.

Clinical signs of mucosal erosions and diarrhea which occur in the acuteform of bovine viral diarrhea have a significant effect on those animalsinfected, but much more costly are animals which are persistentlyinfected. Typically, such animals fail to reach their genetic potential,exhibiting decreased weight gain, increased disease susceptibility andreduced fertility. They shed the virus causing reproductive loss in theunimmunized animals in the herd.

Cows that are exposed to the cytopathic variant of BVDV (45-125 daysgestation) will typically abort the fetus. Earlier exposure to eithervariant leads to early embryonic death. Exposure between days 125-175days of gestation leads to birth defects (such as ocular defects andhydrocephalus) and exposure at greater than 175 days will typically leadto the calf being fully immune at birth.

Therefore, as a consequence of the severity of BRDC and the significanteffect on the livestock industry improvements in vaccination arerequired. Attenuated viruses give better protection than inactivatedviruses because they present more viral antigens to the immune system ofthe host. Another important advantage of the attenuated virus is thepotential to administer it intranasally, i.e. at the site where thefirst multiplication of the wild-type virus occurs after infection.

It has long been recognized that the antigenic variability of BVDV makesit a difficult virus against which to vaccinate. There are twoapproaches which can be taken for BVDV specific vaccination. One is theinduction of neutralizing antibodies which prevent the target virus frominfecting cells. The second is the induction of cell-mediated immunity(CMI) which targets virus infected cells for destruction, thus reducingthe effects of a viral infection. The major neutralizing epitopes ofBVDV are the structural glycoproteins and as a result of immuneselection, these proteins are also the most variable. Thus, designing avaccine based on the glycoproteins requires the inclusion of the mostcommon antigenic types. The non-structural proteins of BVDV aregenerally more conserved as they have a specific enzyme function whichlimits the variation in the protein sequences that can occur.

For a proper BRDC control program, it is necessary to have anefficacious and safe vaccine that can be distinguished from thewild-type virus. Previously developed vaccines using BoHV-1 wereconstructed with deletions to glycoproteins and/or comprised a thymidinekinase deletion mutant. There have been problems with these vaccines asthe thymidine kinase gene is involved in viral replication and lessreplication can lead to less protection due to lower levels ofglycoproteins which are involved in the generation of humoral immunity.

There is a need to develop improved and more efficacious vaccines whichenable control of BRDC and particular pathogens associated therewith.

SUMMARY

Nucleotide and amino acid sequences are referred to by a sequenceidentifier number (SEQ ID NO). The SEQ ID NOs correspond numerically tothe sequence identifiers <400>1 (SEQ ID NO:1), <400>2 (SEQ ID NO:2),etc. A summary of the sequence identifiers is provided in Table 1. Asequence listing is provided after the claims.

Bovine respiratory disease complex (BRDC) represents a significantdisease risk for bovine animals, especially those maintained in confinedenvironments such as feed lots and dairy facilities. Infection bypathogenic agents which are associated with BRDC can spread quickly andcan result in significant morbidity, mortality and loss of production.Taught herein is an improved vaccine carrier comprising the genome froma low virulence strain of BoHV-1 modified to carry genetic materialencoding one or more antigens from bovine pathogens.

Accordingly, enabled herein is a vaccine against at least one antigenfrom a bovine pathogen, the vaccine comprising a bovine herpes virus-1(BoHV-1) genome from a low virulence BoHV-1 having genetic materialencoding the at least one antigen which is heterologous to BoHV-1inserted between two converging BoHV-1 genes wherein the insertion doesnot substantially down-regulate expression of the BoHV-1 genes.

The vaccine has the capacity to be multivalent in respect of stimulatingan immune response to BoHV-1 as well as the antigen associated withanother bovine pathogen such as BVDV, Mycoplasma, Pasteurella, Manhiemiaand Haemophilus. Examples of BVDV antigens include glycoproteins E0 andE2.

In an embodiment, the heterologous genetic material is introduced usingan inducible recombination system such as GET recombination.

Another aspect taught herein is a method for vaccinating a bovine animalagainst at least one antigen from a bovine pathogen, the methodcomprising administering to the bovine animal a humoralimmunity-inducing or cell-mediated immunity-inducing effective amount ofa BoHV-1 genome from a low virulence BoHV-1 having genetic materialencoding the at least one antigen which is heterologous to BoHV-1inserted between two converging BoHV-1 genes wherein the insertion doesnot substantially down-regulate expression of the BoHV-1 genes.

Enabled herein is a method of producing a vaccine against at least oneantigen from a bovine pathogen, the method comprising:

(i) incorporating a BoHV-1 genome from a low virulence BoHV-1 into abacterial artificial chromosome (BAC) vector to form a BoHV-1 pre-vectorBAC construct;

(ii) inserting genetic material encoding the at least one antigen intothe BoHV-1 pre-vector BAC construct via an inducible recombinationsystem to generate a recombinant BoHV-1-BAC (rBoHV-1-BAC) vector;

(iii) transforming and amplifying the rBoHV-1-BAC vector in a bacterialhost; and

(iv) purifying and isolating the rBoHV-1-BAC vector from the bacterialhost and formulating the vector into a vaccine composition.

A method is also provided for vaccinating against bovine respiratorydisease complex (BRDC) in cattle, the method comprising administering tothe cattle a humoral immunity-inducing or cell-mediatedimmunity-inducing effective amount of a bovine herpes virus-1 (BoHV-1)genome from a low virulence BoHV-1 having genetic material encoding theat least one antigen which is heterologous to BoHV-1 inserted betweentwo converging BoHV-1 genes wherein the insertion does not substantiallydown-regulate expression of the BoHV-1 genes.

The present disclosure enables a use of a BoHV-1 genome from a lowvirulence BoHV-1 having genetic material encoding the at least oneantigen which is heterologous to BoHV-1 inserted between two convergingBoHV-1 genes wherein the insertion does not substantially down-regulateexpression of the BoHV-1 genes in the manufacture of a medicament in thevaccination of cattle against a bovine pathogen.

Enabled herein is a method of producing a vaccine against at least oneantigen from a bovine pathogen, the method comprising:

(i) incorporating a BoHV-1 genome from a low virulence BoHV-1 into abacterial artificial chromosome (BAC) vector to form a BoHV-1 pre-vectorBAC construct;

(ii) inserting genetic material encoding the at least one antigen intothe BoHV-1 pre-vector BAC construct via an inducible recombinationsystem to generate a recombinant BoHV-1-BAC (rBoHV-1-BAC) vector;

(iii) transforming and amplifying the rBoHV-1-BAC vector in a bacterialhost; and

(iv) purifying and isolating the rBoHV-1-BAC vector from the bacterialhost and formulating the vector into a vaccine composition.

A BoHV-1 genome from a low virulence BoHV-1 which when expressedproduces an antigen to which an immune response is capable of beinggenerated, the BoHV-1 genome further comprising genetic materialencoding at least one other antigen heterologous to BoHV-1 insertedbetween two converging BoHV-1 genes wherein the insertion does notsubstantially down-regulate expression of the BoHV-1 genes and whereinthe heterologous antigen induces an immune response.

Hence, taught herein is a vaccine vector comprising a BoHV-1 genome froma low virulence BoHV-1 having genetic material encoding the at least oneantigen which is heterologous to BoHV-1 inserted between two convergingBoHV-1 genes wherein the insertion does not substantially down-regulateexpression of the BoHV-1 genes.

A polyvalent vaccine vector is enabled herein comprising:

(1) a first valency comprising a BoHV-1 genome from a low virulenceBoHV-1; and

(2) a second valency comprising genetic material encoding at least oneantigen which is heterologous to the BoHV-1 inserted between twoconverging BoHV-1 genes wherein the insertion does not substantiallydown-regulate expression of the BoHV-1 genes;

wherein the first and second valencies produce two or more antigens towhich an immune response is generated in a bovine host.

Further enabled herein is a BoHV-1 vaccine vector comprising a BoHV-1genome derived from BoHV-1 strain V155 having heterologous geneticmaterial encoding at least one antigen from a bovine pathogen insertedinto a site on the BoHV-1 genome selected from nucleotides 16600 to16700; 22400 to 22500; 40,700 to 40,800; 58,000 to 59,000; 67,000 to68,000; 74,000 to 76,000; 84,000 to 85,000; 90,000 to 91,000; and 96,000to 97,000 of BoHV-1 reference sequence GenBank Accession No. AJ004801 orat a functionally equivalent site in another BoHV-1. For examples, referto Table 2.

Pharmaceutical compositions, treatment and vaccination protocols arealso taught the present disclosure as are business methods formanagement of confined or herded bovine animals.

A summary of insertion sites into the BoHV-1 genome between converginggenes is provided in Table 2. Sequence co-ordinates refer to the BoHV-1reference sequence deposited with GenBank Accession No. AJ004801.

TABLE 1 SEQUENCE ID NO: DESCRIPTION 1 Nucleotide sequence of Tkleft 5′primer 2 Nucleotide sequence of Tkleft 3′ primer 3 Nucleotide sequenceof Tkright 5′ primer 4 Nucleotide sequence of Tkright 3′ primer 5Nucleotide sequence of ChloramF primer 6 Nucleotide sequence of ChloramRprimer 7 Nucleotide sequence of gE-KanF primer 8 Nucleotide sequence ofgE-KanR primer 9 Nucleotide sequence of BHV1.3 primer 10 Nucleotidesequence of BHV1.6 primer 11 Nucleotide sequence of KanR fwd primer 12Nucleotide sequence of KanR rev primer

TABLE 2 List of insertion sites¹ into the bovine herpesvirus 1 (BoHV-1)genome between converging genes Insertion Site Start End CommentsInsertion Site 1 16600 16612 Convergent genes UL46 & UL44 Insertion Site2 22449 22493 Convergent genes UL41 & UL40 Insertion Site 3 40734 40768Convergent genes UL36 & UL35 Insertion Site 4 58229 58563 Convergentgenes UL27 & UL26 Insertion Site 5 67037 67091 Convergent genes UL22 &UL21 Insertion Site 6 74994 75041 Convergent genes UL19 & UL15 InsertionSite 7 84496 84528 Convergent genes UL11 & UL10 Insertion Site 8 9073290760 Convergent genes UL8 & UL7 Insertion Site 9 96870 96882 Convergentgenes UL4 & UL3.6 ¹Sequence co-ordinates refer to the BoHV-1 referencesequence with GenBank Accession AJ004801 or its equivalent

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Graphical representations showing a comparison of the virusyield of various mammalian-derived cell-lines infected with eitherparent Bovine herpesvirus-1 or recombinant Bovine herpesvirus carryglycoprotein E2 from bovine viral diarrhea virus at 24 hrspost-infection. (A) Cells of primate origin; (B) Cells of bovine origin;(C) Cells of rabbit and small ruminant origin. Yield of virus wasdetermined by real-time PCR amplification performed in triplicate.

DETAILED DESCRIPTION

Throughout this specification, unless the context requires otherwise,the word “comprise” or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated element or integeror method step or group of elements or integers or method steps but notthe exclusion of any element or integer or method step or group ofelements or integers or method steps.

As used in the subject specification, the singular forms “a”, “an” and“the” include plural aspects unless the context clearly dictatesotherwise. Thus, for example, reference to “a virus” includes a singlevirus, as well as two or more viruses; reference to “an antigen”includes a single antigen, as well as two or more antigens; reference to“the disclosure” includes a single or multiple aspects taught therein.

The present disclosure teaches a recombinant vaccine vector in the formof BoHV-1 from a low virulence strain of the virus. In an embodiment,the low virulence strain is referred to as BoHV-1 V155 (Snowden (1964)Australian Veterinary Journal 40:277-288). The recombinant vaccinevector is used as a vehicle to express proteins heterologous to BoHV-1from bovine pathogens to which an immune response is sought. The genomeportion of the BoHV-1 vector itself may also express proteins whichinduce an anti-BoHV-1 immune response. The immune response in bovineanimals is regarded, in an embodiment, as a protective immune responsein that the immune response targets the protein on or produced by apathogen and this facilitates a reduction in infection, colonizationand/or symptoms of disease and/or transmission of pathogens and/oroutcomes of infection such as morbidity or mortality. The immuneresponse may be humoral and/or cell-mediated.

In an embodiment, the BoHV-1 vector is genetically manipulated to insertgenes from a bovine pathogen in between convergent genes on the BoHV-1genome. The insertion does not, in an embodiment, substantially decreaseexpression of the two flanking BoHV-1 genes nor any other gene in theBoHV-1 genome. Upon infection of cells of a bovine animal with therecombinant vaccine vector, the pathogen gene(s) is/are expressed toform a protein antigen(s) and an immune response elicited against theone or more pathogen antigens. As indicated above, the BoHV-1 vectoritself provides a target for immunological stimulation against BoHV-1.Hence, the present disclosure teaches the facilitation of a dual vaccineapproach based on the stimulation of an immune response against BoHV-1and an immune response against a heterologous protein geneticallyengineered to be expressed by the BoHV-1 vaccine vector.

Accordingly, enabled herein is a vaccine against at least one antigenfrom a bovine pathogen, the vaccine comprising a BoHV-1 genome from alow virulence BoHV-1 having genetic material encoding the at least oneantigen which is heterologous to BoHV-1 inserted between two convergingBoHV-1 genes wherein the insertion does not substantially down-regulateexpression of the BoHV-1 genes.

The vaccine enables expression of the heterologous antigen to facilitatethe stimulation of an immune response against the antigen. In addition,the BoHV-1 vector itself may facilitate an immune response to a BoHV-1protein. The use of a low virulence BoHV-1 rather than an inactivated orattenuated strain improves its ability to infect, replicate and producea non-pathogenic infection and an effective immune response againstBoHV-1 and any heterologous antigens.

Taught herein is a multivalent vaccine against two or more antigens froma bovine pathogen, the vaccine comprising a BoHV-1 genome from a lowvirulence BoHV-1 which when expressed produces an antigen to which animmune response is generated, the BoHV-1 genome further comprisinggenetic material encoding at least one other antigen heterologous toBoHV-1 inserted between two converging BoHV-1 genes wherein theinsertion does not substantially down-regulate expression of the BoHV-1genes and wherein the heterologous antigen induces an immune response.

The terms “multivalent” and “polyvalent” may be used interchangeably todescribe this aspect enabled herein.

The vaccine taught herein is also considered a vaccine vector.

Accordingly, another aspect enabled herein is a vaccine vectorcomprising a BoHV-1 genome from a low virulence BoHV-1 having geneticmaterial encoding the at least one antigen which is heterologous toBoHV-1 inserted between two converging BoHV-1 genes wherein theinsertion does not substantially down-regulate expression of the BoHV-1genes.

Another aspect enabled herein is a polyvalent vaccine vector comprising:

(1) a first valency comprising a BoHV-1 genome from a low virulenceBoHV-1; and

(2) a second valency comprising genetic material encoding at least oneantigen which is heterologous to the BoHV-1 inserted between twoconverging BoHV-1 genes wherein the insertion does not substantiallydown-regulate expression of the BoHV-1 genes;

wherein the first and second valencies produce two or more antigens towhich an immune response is generated in a bovine host.

The term “substantially” in relation to the down-regulation means thatthere is either no down-regulation of expression or there is only aminor reduction in expression. By “minor” means that from a functionalperspective, any change in expression does not adversely affect thefunctioning of the virus.

As indicated above, the “immune response” may be a humoral immuneresponse and/or a cell-mediated immune response.

The vaccine enabled herein permits treatment or prophylaxis of bovinerespiratory disease complex (BRDC) which is a particularly prevalent indisease in lot or herded cattle. By “lot cattle” includes cattleconfined for feeding, rearing or dairying purposes. BRDC is amulti-factorial disease. Typically, a bovine animal is infected with oneor more of BoHV-1, BVDV, Bovine parainfluenza 3 and/or Bovinerespiratory syncytial virus. This often leads to secondary viral ormicrobial infection and results in conditions such as pneumonia.

Microbial pathogens contemplated herein include Mycoplasma sp,Salmonella sp, Pasteurella sp, Manhiemia sp and Haemophilus sp such asMycoplasma bovis, Pasteurella multocida, Manhiemia haemolytica andHaemophilus somnus. Genetic material encoding antigens from any or allof these or other bacteria may be used in the BoHV-1 vaccine vector.BVDV antigens include glycoproteins E0 and E2.

Accordingly, the instant disclosure enables a method of vaccinating abovine animal against at least one antigen from a bovine pathogen, themethod comprising administering to the bovine animal, a humoralimmunity-inducing or cell-mediated immunity-inducing effective amount ofa BoHV-1 genome from a low virulence BoHV-1 having genetic materialencoding the at least one antigen which is heterologous to BoHV-1inserted between two converging BoHV-1 genes wherein the insertion doesnot substantially down-regulate expression of the BoHV-1 genes.

The present disclosure teaches a method for vaccinating against BRDC incattle, the method comprising administering to the cattle, a humoralimmunity-inducing or cell-mediated immunity-inducing effective amount ofa BoHV-1 genome from a low virulence BoHV-1 having genetic materialencoding the at least one antigen which is heterologous to BoHV-1inserted between two converging BoHV-1 genes wherein the insertion doesnot substantially down-regulate expression of the BoHV-1 genes.

Genetic manipulation of the BoHV-1 vaccine vector to insert heterologousnucleic acid material is generally by an inducible recombination system.In an embodiment, the inducible recombination system is GETrecombination which utilizes transient expression of recE and recT toenable homologous recombination in Escherichia coli (see Orford et al.Nucleic Acids Research 28(18):e84; Mahoney et al. (2002) Journal ofVirology 76(13):6660-6668; Narayanan et al. (1999) Gene therapy6:442-447; Schumacher et al. (2000) Journal of Virology 74:11088-11098).

In an embodiment, the heterologous genetic material is inserted betweenthe polyadenylation signals of two converging genes at a site selectedfrom 16600 to 16700 and 22400 to 22500 of BoHV-1 reference sequenceGenBank Accession No. AJ004801 or at a functionally equivalent site inanother BoHV-1. In an embodiment, the heterologous genetic material isinserted at a site selected from between nucleotides 16600 to 16700 and22400 to 22493 based on sequence coordinates of BoHV-1 referencesequence GenBank Accession No. AJ004801 or its equivalent. Reference to“16600 to 16700” includes 16600, 16601, 16602, 16603, 16604, 16605,16606, 16607, 16608, 16609, 16610, 16611, 16612, 16613, 16614, 16615,16616, 16617, 16618, 16619, 16620, 16621, 16622, 16623, 16624, 16625,16626, 16627, 16628, 16629, 16630, 16631, 16632, 16633, 16634, 16635,16636, 16637, 16638, 16639, 16640, 16641, 16642, 16643, 16644, 16645,16646, 16647, 16648, 16649, 16650, 16651, 16652, 16653, 16654, 16655,16656, 16657, 16658, 16659, 16660, 16661, 16662, 16663, 16664, 16665,16667, 16668, 16669, 16670, 16671, 16672, 16673, 16674, 16675, 16676,16677, 16678, 16679, 16680, 16681, 16682, 16683, 16684, 16685, 16686,16687, 16688, 16689, 16690, 16691, 16692, 16693, 16694, 16695, 16696,16697, 16698, 16699 and 16700. Reference to “22400 to 22500” includes22400, 22401, 22402, 22403, 22404, 22405, 22406, 22407, 22408, 22409,22410, 22411, 22412, 22413, 22414, 22415, 22416, 22417, 22418, 22419,22420, 22421, 22422, 22423, 22424, 22425, 22426, 22427, 22428, 22429,22430, 22431, 22432, 22433, 22434, 22435, 22436, 22437, 22438, 22439,22440, 22441, 22442, 22443, 22444, 22445, 22446, 22447, 22448, 22449,22450, 22451, 22452, 22453, 22454, 22455, 22456, 22457, 22458, 22459,22460, 22461, 22462, 22463, 22464, 22465, 22466, 22467, 22468, 22469,22470, 22471, 22472, 22473, 22474, 22475, 22476, 22477, 22478, 22479,22480, 22481, 22482, 22483, 22484, 22485, 22486, 22487, 22488, 22489,22490, 22491, 22492, 22493, 22494, 22495, 22496, 22497, 22498, 22499 and22500.

Other sites include within the range 40,700 to 40,800; which encompassessites 40,700, 40,701, 40,702, 40,703, 40,704, 40,705, 40,706, 40,707,40,708, 40,709, 40,710, 40,711, 40,712, 40,713, 40,714, 40,715, 40,716,40,717, 40,718, 40,719, 40,720, 40,721, 40,722, 40,723, 40,724, 40,725,40,726, 40,727, 40,728, 40,729, 40,730, 40,731, 40,732, 40,733, 40,734,40,735, 40,736, 40,737, 40,738, 40,739, 40,740, 40,741, 40,742, 40,743,40,744, 40,745, 40,746, 40,747, 40,748, 40,749, 40,750, 40,751, 40,752,40,753, 40,754, 40,755, 40,756, 40,757, 40,758, 40,759, 40,760, 40,761,40,762, 40,763, 40,764, 40,765, 40,766, 40,767, 40,768, 40,769, 40,770,40,771, 40,772, 40,773, 40,774, 40,775, 40,776, 40,777, 40,778, 40,779,40,780, 40,781, 40,782, 40,783, 40,784, 40,785, 40,786, 40,787, 40,788,40,789, 40,790, 40,791, 40,792, 40,793, 40,794, 40,795, 40,796, 40,797,40,798, 40,799, 40,800; 58,000 to 59,000 include 58,001, 58,002, 58,003,58,004, 58,005, 58,006, 58,007, 58,008, 58,009, 58,010, 58,011, 58,012,58,013, 58,014, 58,015, 58,016, 58,017, 58,018, 58,019, 58,020, 58,021,58,022, 58,023, 58,024, 58,025, 58,026, 58,027, 58,028, 58,029, 58,030,58,031, 58,032, 58,033, 58,034, 58,035, 58,036, 58,037, 58,038, 58,039,58,040, 58,041, 58,042, 58,043, 58,044, 58,045, 58,046, 58,047, 58,048,58,049, 58,050, 58,051, 58,052, 58,053, 58,054, 58,055, 58,056, 58,057,58,058, 58,059, 58,060, 58,061, 58,062, 58,063, 58,064, 58,065, 58,066,58,067, 58,068, 58,069, 58,070, 58,071, 58,072, 58,073, 58,074, 58,075,58,076, 58,077, 58,078, 58,079, 58,080, 58,081, 58,082, 58,083, 58,084,58,085, 58,086, 58,087, 58,088, 58,089, 58,090, 58,091, 58,092, 58,093,58,094, 58,095, 58,096, 58,097, 58,098, 58,099, 58,100, 58,101, 58,102,58,103, 58,104, 58,105, 58,106, 58,107, 58,110, 58,111, 58,112, 58,113,58,114, 58,115, 58,116, 58,117, 58,118, 58,119, 58,120, 58,121, 58,122,58,123, 58,124, 58,125, 58,126, 58,127, 58,128, 58,129, 58,130, 58,131,58,132, 58,133, 58,134, 58,135, 58,136, 58,137, 58,138, 58,139, 58,140,58,141, 58,142, 58,143, 58,144, 58,145, 58,146, 58,147, 58,148, 58,149,58,150, 58,151, 58,152, 58,153, 58,154, 58,155, 58,156, 58,157, 58,158,58,159, 58,160, 58,161, 58,162, 58,163, 58,164, 58,165, 58,166, 58,167,58,168, 58,169, 58,170, 58,171, 58,172, 58,173, 58,174, 58,175, 58,176,58,177, 58,178, 58,179, 58,180, 58,181, 58,182, 58,183, 58,184, 58,185,58,186, 58,187, 58,188, 58,189, 58,190, 58,191, 58,192, 58,193, 58,194,58,195, 58,196, 58,197, 58,198, 58,199, 58,200, 58,201, 58,202, 58,203,58,204, 58,205, 58,206, 58,207, 58,208, 58,209, 58,210, 58,211, 58,212,58,213, 58,214, 58,215, 58,216, 58,217, 58,218, 58,219, 58,220, 58,221,58,222, 58,223, 58,224, 58,225, 58,226, 58,227, 58,228, 58,229, 58,230,58,231, 58,232, 58,233, 58,234, 58,235, 58,236, 58,237, 58,238, 58,239,58,240, 58,241, 58,242, 58,243, 58,244, 58,245, 58,246, 58,247, 58,248,58,249, 58,250, 58,251, 58,252, 58,253, 58,254, 58,255, 58,256, 58,257,58,258, 58,259, 58,260, 58,261, 58,262, 58,263, 58,264, 58,265, 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75,539,75,540, 75,541, 75,542, 75,543, 75,544, 75,545, 75,546, 75,547, 75,548,75,549, 75,550, 75,551, 75,552, 75,553, 75,554, 75,555, 75,556, 75,557,75,575, 75,559, 75,560, 75,561, 75,562, 75,563, 75,564, 75,565, 75,566,75,575, 75,568, 75,569, 75,570, 75,571, 75,572, 75,573, 75,575, 75,575,75,576, 75,577, 75,578, 75,579, 75,580, 75,581, 75,582, 75,583, 75,584,75,585, 75,586, 75,587, 75,588, 75,589, 75,590, 75,591, 75,592, 75,593,75,594, 75,595, 75,596, 75,597, 75,598, 75,599, 75,600, 75,601, 75,602,75,603, 75,604, 75,605, 75,606, 75,607, 75,608, 75,609, 75,610, 75,611,75,612, 75,613, 75,614, 75,615, 75,616, 75,617, 75,618, 75,619, 75,620,75,621, 75,622, 75,623, 75,624, 75,625, 75,626, 75,627, 75,628, 75,629,75,630, 75,631, 75,632, 75,633, 75,634, 75,635, 75,636, 75,637, 75,638,75,639, 75,640, 75,641, 75,642, 75,643, 75,644, 75,645, 75,646, 75,647,75,648, 75,649, 75,650, 75,651, 75,652, 75,653, 75,654, 75,655, 75,656,75,657, 75,675, 75,659, 75,660, 75,661, 75,662, 75,663, 75,664, 75,665,75,666, 75,675, 75,668, 75,669, 75,670, 75,671, 75,672, 75,673, 75,675,75,675, 75,676, 75,677, 75,678, 75,679, 75,680, 75,681, 75,682, 75,683,75,684, 75,685, 75,686, 75,687, 75,688, 75,689, 75,690, 75,691, 75,692,75,693, 75,694, 75,695, 75,696, 75,697, 75,698, 75,699, 75,700, 75,701,75,702, 75,703, 75,704, 75,705, 75,706, 75,707, 75,708, 75,709, 75,710,75,711, 75,712, 75,713, 75,714, 75,715, 75,716, 75,717, 75,718, 75,719,75,720, 75,721, 75,722, 75,723, 75,724, 75,725, 75,726, 75,727, 75,728,75,729, 75,730, 75,731, 75,732, 75,733, 75,734, 75,735, 75,736, 75,737,75,738, 75,739, 75,740, 75,741, 75,742, 75,743, 75,744, 75,745, 75,746,75,747, 75,748, 75,749, 75,750, 75,751, 75,752, 75,753, 75,754, 75,755,75,756, 75,757, 75,775, 75,759, 75,760, 75,761, 75,762, 75,763, 75,764,75,765, 75,766, 75,775, 75,768, 75,769, 75,770, 75,771, 75,772, 75,773,75,775, 75,775, 75,776, 75,777, 75,778, 75,779, 75,780, 75,781, 75,782,75,783, 75,784, 75,785, 75,786, 75,787, 75,788, 75,789, 75,790, 75,791,75,792, 75,793, 75,794, 75,795, 75,796, 75,797, 75,798, 75,799, 75,800,75,801, 75,802, 75,803, 75,804, 75,805, 75,806, 75,807, 75,808, 75,809,75,810, 75,811, 75,812, 75,813, 75,814, 75,815, 75,816, 75,817, 75,818,75,819, 75,820, 75,821, 75,822, 75,823, 75,824, 75,825, 75,826, 75,827,75,828, 75,829, 75,830, 75,831, 75,832, 75,833, 75,834, 75,835, 75,836,75,837, 75,838, 75,839, 75,840, 75,841, 75,842, 75,843, 75,844, 75,845,75,846, 75,847, 75,848, 75,849, 75,850, 75,851, 75,852, 75,853, 75,854,75,855, 75,856, 75,857, 75,875, 75,859, 75,860, 75,861, 75,862, 75,863,75,864, 75,865, 75,866, 75,875, 75,868, 75,869, 75,870, 75,871, 75,872,75,873, 75,875, 75,875, 75,876, 75,877, 75,878, 75,879, 75,880, 75,881,75,882, 75,883, 75,884, 75,885, 75,886, 75,887, 75,888, 75,889, 75,890,75,891, 75,892, 75,893, 75,894, 75,895, 75,896, 75,897, 75,898, 75,899,75,900, 75,901, 75,902, 75,903, 75,904, 75,905, 75,906, 75,907, 75,908,75,909, 75,910, 75,911, 75,912, 75,913, 75,914, 75,915, 75,916, 75,917,75,918, 75,919, 75,920, 75,921, 75,922, 75,923, 75,924, 75,925, 75,926,75,927, 75,928, 75,929, 75,930, 75,931, 75,932, 75,933, 75,934, 75,935,75,936, 75,937, 75,938, 75,939, 75,940, 75,941, 75,942, 75,943, 75,944,75,945, 75,946, 75,947, 75,948, 75,949, 75,950, 75,951, 75,952, 75,953,75,954, 75,955, 75,956, 75,957, 75,975, 75,959, 75,960, 75,961, 75,962,75,963, 75,964, 75,965, 75,966, 75,975, 75,968, 75,969, 75,970, 75,971,75,972, 75,973, 75,975, 75,975, 75,976, 75,977, 75,978, 75,979, 75,980,75,981, 75,982, 75,983, 75,984, 75,985, 75,986, 75,987, 75,988, 75,989,75,990, 75,991, 75,992, 75,993, 75,994, 75,995, 75,996, 75,997, 75,998,75,999 or 76,000; 84,000 to 85,000 include 84,001, 84,002, 84,003,84,004, 84,005, 84,006, 84,007, 84,008, 84,009, 84,010, 84,011, 84,012,84,013, 84,014, 84,015, 84,016, 84,017, 84,018, 84,019, 84,020, 84,021,84,022, 84,023, 84,024, 84,025, 84,026, 84,027, 84,028, 84,029, 84,030,84,031, 84,032, 84,033, 84,034, 84,035, 84,036, 84,037, 84,038, 84,039,84,040, 84,041, 84,042, 84,043, 84,044, 84,045, 84,046, 84,047, 84,048,84,049, 84,050, 84,051, 84,052, 84,053, 84,054, 84,055, 84,056, 84,057,84,084, 84,059, 84,060, 84,061, 84,062, 84,063, 84,064, 84,065, 84,066,84,084, 84,068, 84,069, 84,070, 84,071, 84,072, 84,073, 84,084, 84,084,84,076, 84,077, 84,078, 84,079, 84,080, 84,081, 84,082, 84,083, 84,084,84,085, 84,086, 84,087, 84,088, 84,089, 84,090, 84,091, 84,092, 84,093,84,094, 84,095, 84,096, 84,097, 84,098, 84,099, 84,100, 84,101, 84,102,84,103, 84,104, 84,105, 84,106, 84,107, 84,110, 84,111, 84,112, 84,113,84,114, 84,115, 84,116, 84,117, 84,118, 84,119, 84,120, 84,121, 84,122,84,123, 84,124, 84,125, 84,126, 84,127, 84,128, 84,129, 84,130, 84,131,84,132, 84,133, 84,134, 84,135, 84,136, 84,137, 84,138, 84,139, 84,140,84,141, 84,142, 84,143, 84,144, 84,145, 84,146, 84,147, 84,148, 84,149,84,150, 84,151, 84,152, 84,153, 84,154, 84,155, 84,156, 84,157, 84,184,84,159, 84,160, 84,161, 84,162, 84,163, 84,164, 84,165, 84,166, 84,184,84,168, 84,169, 84,170, 84,171, 84,172, 84,173, 84,184, 84,184, 84,176,84,177, 84,178, 84,179, 84,180, 84,181, 84,182, 84,183, 84,184, 84,185,84,186, 84,187, 84,188, 84,189, 84,190, 84,191, 84,192, 84,193, 84,194,84,195, 84,196, 84,197, 84,198, 84,199, 84,200, 84,201, 84,202, 84,203,84,204, 84,205, 84,206, 84,207, 84,208, 84,209, 84,210, 84,211, 84,212,84,213, 84,214, 84,215, 84,216, 84,217, 84,218, 84,219, 84,220, 84,221,84,222, 84,223, 84,224, 84,225, 84,226, 84,227, 84,228, 84,229, 84,230,84,231, 84,232, 84,233, 84,234, 84,235, 84,236, 84,237, 84,238, 84,239,84,240, 84,241, 84,242, 84,243, 84,244, 84,245, 84,246, 84,247, 84,248,84,249, 84,250, 84,251, 84,252, 84,253, 84,254, 84,255, 84,256, 84,257,84,284, 84,259, 84,260, 84,261, 84,262, 84,263, 84,264, 84,265, 84,266,84,284, 84,268, 84,269, 84,270, 84,271, 84,272, 84,273, 84,284, 84,284,84,276, 84,277, 84,278, 84,279, 84,280, 84,281, 84,282, 84,283, 84,284,84,285, 84,286, 84,287, 84,288, 84,289, 84,290, 84,291, 84,292, 84,293,84,294, 84,295, 84,296, 84,297, 84,298, 84,299, 84,300, 84,301, 84,302,84,303, 84,304, 84,305, 84,306, 84,307, 84,308, 84,309, 84,310, 84,311,84,312, 84,313, 84,314, 84,315, 84,316, 84,317, 84,318, 84,319, 84,320,84,321, 84,322, 84,323, 84,324, 84,325, 84,326, 84,327, 84,328, 84,329,84,330, 84,331, 84,332, 84,333, 84,334, 84,335, 84,336, 84,337, 84,338,84,339, 84,340, 84,341, 84,342, 84,343, 84,344, 84,345, 84,346, 84,347,84,348, 84,349, 84,350, 84,351, 84,352, 84,353, 84,354, 84,355, 84,356,84,357, 84,384, 84,359, 84,360, 84,361, 84,362, 84,363, 84,364, 84,365,84,366, 84,384, 84,368, 84,369, 84,370, 84,371, 84,372, 84,373, 84,384,84,384, 84,376, 84,377, 84,378, 84,379, 84,380, 84,381, 84,382, 84,383,84,384, 84,385, 84,386, 84,387, 84,388, 84,389, 84,390, 84,391, 84,392,84,393, 84,394, 84,395, 84,396, 84,397, 84,398, 84,399, 84,400, 84,401,84,402, 84,403, 84,404, 84,405, 84,406, 84,407, 84,408, 84,409, 84,410,84,411, 84,412, 84,413, 84,414, 84,415, 84,416, 84,417, 84,418, 84,419,84,420, 84,421, 84,422, 84,423, 84,424, 84,425, 84,426, 84,427, 84,428,84,429, 84,430, 84,431, 84,432, 84,433, 84,434, 84,435, 84,436, 84,437,84,438, 84,439, 84,440, 84,441, 84,442, 84,443, 84,444, 84,445, 84,446,84,447, 84,448, 84,449, 84,450, 84,451, 84,452, 84,453, 84,454, 84,455,84,456, 84,457, 84,484, 84,459, 84,460, 84,461, 84,462, 84,463, 84,464,84,465, 84,466, 84,484, 84,468, 84,469, 84,470, 84,471, 84,472, 84,473,84,484, 84,484, 84,476, 84,477, 84,478, 84,479, 84,480, 84,481, 84,482,84,483, 84,484, 84,485, 84,486, 84,487, 84,488, 84,489, 84,490, 84,491,84,492, 84,493, 84,494, 84,495, 84,496, 84,497, 84,498, 84,499, 84,500,84,501, 84,502, 84,503, 84,504, 84,505, 84,506, 84,507, 84,508, 84,509,84,510, 84,511, 84,512, 84,513, 84,514, 84,515, 84,516, 84,517, 84,518,84,519, 84,520, 84,521, 84,522, 84,523, 84,524, 84,525, 84,526, 84,527,84,528, 84,529, 84,530, 84,531, 84,532, 84,533, 84,534, 84,535, 84,536,84,537, 84,538, 84,539, 84,540, 84,541, 84,542, 84,543, 84,544, 84,545,84,546, 84,547, 84,548, 84,549, 84,550, 84,551, 84,552, 84,553, 84,554,84,555, 84,556, 84,557, 84,584, 84,559, 84,560, 84,561, 84,562, 84,563,84,564, 84,565, 84,566, 84,584, 84,568, 84,569, 84,570, 84,571, 84,572,84,573, 84,584, 84,584, 84,576, 84,577, 84,578, 84,579, 84,580, 84,581,84,582, 84,583, 84,584, 84,585, 84,586, 84,587, 84,588, 84,589, 84,590,84,591, 84,592, 84,593, 84,594, 84,595, 84,596, 84,597, 84,598, 84,599,84,600, 84,601, 84,602, 84,603, 84,604, 84,605, 84,606, 84,607, 84,608,84,609, 84,610, 84,611, 84,612, 84,613, 84,614, 84,615, 84,616, 84,617,84,618, 84,619, 84,620, 84,621, 84,622, 84,623, 84,624, 84,625, 84,626,84,627, 84,628, 84,629, 84,630, 84,631, 84,632, 84,633, 84,634, 84,635,84,636, 84,637, 84,638, 84,639, 84,640, 84,641, 84,642, 84,643, 84,644,84,645, 84,646, 84,647, 84,648, 84,649, 84,650, 84,651, 84,652, 84,653,84,654, 84,655, 84,656, 84,657, 84,684, 84,659, 84,660, 84,661, 84,662,84,663, 84,664, 84,665, 84,666, 84,684, 84,668, 84,669, 84,670, 84,671,84,672, 84,673, 84,684, 84,684, 84,676, 84,677, 84,678, 84,679, 84,680,84,681, 84,682, 84,683, 84,684, 84,685, 84,686, 84,687, 84,688, 84,689,84,690, 84,691, 84,692, 84,693, 84,694, 84,695, 84,696, 84,697, 84,698,84,699, 84,700, 84,701, 84,702, 84,703, 84,704, 84,705, 84,706, 84,707,84,708, 84,709, 84,710, 84,711, 84,712, 84,713, 84,714, 84,715, 84,716,84,717, 84,718, 84,719, 84,720, 84,721, 84,722, 84,723, 84,724, 84,725,84,726, 84,727, 84,728, 84,729, 84,730, 84,731, 84,732, 84,733, 84,734,84,735, 84,736, 84,737, 84,738, 84,739, 84,740, 84,741, 84,742, 84,743,84,744, 84,745, 84,746, 84,747, 84,748, 84,749, 84,750, 84,751, 84,752,84,753, 84,754, 84,755, 84,756, 84,757, 84,784, 84,759, 84,760, 84,761,84,762, 84,763, 84,764, 84,765, 84,766, 84,784, 84,768, 84,769, 84,770,84,771, 84,772, 84,773, 84,784, 84,784, 84,776, 84,777, 84,778, 84,779,84,780, 84,781, 84,782, 84,783, 84,784, 84,785, 84,786, 84,787, 84,788,84,789, 84,790, 84,791, 84,792, 84,793, 84,794, 84,795, 84,796, 84,797,84,798, 84,799, 84,800, 84,801, 84,802, 84,803, 84,804, 84,805, 84,806,84,807, 84,808, 84,809, 84,810, 84,811, 84,812, 84,813, 84,814, 84,815,84,816, 84,817, 84,818, 84,819, 84,820, 84,821, 84,822, 84,823, 84,824,84,825, 84,826, 84,827, 84,828, 84,829, 84,830, 84,831, 84,832, 84,833,84,834, 84,835, 84,836, 84,837, 84,838, 84,839, 84,840, 84,841, 84,842,84,843, 84,844, 84,845, 84,846, 84,847, 84,848, 84,849, 84,850, 84,851,84,852, 84,853, 84,854, 84,855, 84,856, 84,857, 84,884, 84,859, 84,860,84,861, 84,862, 84,863, 84,864, 84,865, 84,866, 84,884, 84,868, 84,869,84,870, 84,871, 84,872, 84,873, 84,884, 84,884, 84,876, 84,877, 84,878,84,879, 84,880, 84,881, 84,882, 84,883, 84,884, 84,885, 84,886, 84,887,84,888, 84,889, 84,890, 84,891, 84,892, 84,893, 84,894, 84,895, 84,896,84,897, 84,898, 84,899, 84,900, 84,901, 84,902, 84,903, 84,904, 84,905,84,906, 84,907, 84,908, 84,909, 84,910, 84,911, 84,912, 84,913, 84,914,84,915, 84,916, 84,917, 84,918, 84,919, 84,920, 84,921, 84,922, 84,923,84,924, 84,925, 84,926, 84,927, 84,928, 84,929, 84,930, 84,931, 84,932,84,933, 84,934, 84,935, 84,936, 84,937, 84,938, 84,939, 84,940, 84,941,84,942, 84,943, 84,944, 84,945, 84,946, 84,947, 84,948, 84,949, 84,950,84,951, 84,952, 84,953, 84,954, 84,955, 84,956, 84,957, 84,984, 84,959,84,960, 84,961, 84,962, 84,963, 84,964, 84,965, 84,966, 84,984, 84,968,84,969, 84,970, 84,971, 84,972, 84,973, 84,984, 84,984, 84,976, 84,977,84,978, 84,979, 84,980, 84,981, 84,982, 84,983, 84,984, 84,985, 84,986,84,987, 84,988, 84,989, 84,990, 84,991, 84,992, 84,993, 84,994, 84,995,84,996, 84,997, 84,998, 84,999 or 85,000; 90,000 to 91,000 include90,001, 90,002, 90,003, 90,004, 90,005, 90,006, 90,007, 90,008, 90,009,90,010, 90,011, 90,012, 90,013, 90,014, 90,015, 90,016, 90,017, 90,018,90,019, 90,020, 90,021, 90,022, 90,023, 90,024, 90,025, 90,026, 90,027,90,028, 90,029, 90,030, 90,031, 90,032, 90,033, 90,034, 90,035, 90,036,90,037, 90,038, 90,039, 90,040, 90,041, 90,042, 90,043, 90,044, 90,045,90,046, 90,047, 90,048, 90,049, 90,050, 90,051, 90,052, 90,053, 90,054,90,055, 90,056, 90,057, 90,090, 90,059, 90,060, 90,061, 90,062, 90,063,90,064, 90,065, 90,066, 90,090, 90,068, 90,069, 90,070, 90,071, 90,072,90,073, 90,090, 90,090, 90,076, 90,077, 90,078, 90,079, 90,080, 90,081,90,082, 90,083, 90,090, 90,085, 90,086, 90,087, 90,088, 90,089, 90,090,90,091, 90,092, 90,093, 90,094, 90,095, 90,096, 90,097, 90,098, 90,099,90,100, 90,101, 90,102, 90,103, 90,104, 90,105, 90,106, 90,107, 90,110,90,111, 90,112, 90,113, 90,114, 90,115, 90,116, 90,117, 90,118, 90,119,90,120, 90,121, 90,122, 90,123, 90,124, 90,125, 90,126, 90,127, 90,128,90,129, 90,130, 90,131, 90,132, 90,133, 90,134, 90,135, 90,136, 90,137,90,138, 90,139, 90,140, 90,141, 90,142, 90,143, 90,144, 90,145, 90,146,90,147, 90,148, 90,149, 90,150, 90,151, 90,152, 90,153, 90,154, 90,155,90,156, 90,157, 90,190, 90,159, 90,160, 90,161, 90,162, 90,163, 90,164,90,165, 90,166, 90,190, 90,168, 90,169, 90,170, 90,171, 90,172, 90,173,90,190, 90,190, 90,176, 90,177, 90,178, 90,179, 90,180, 90,181, 90,182,90,183, 90,190, 90,185, 90,186, 90,187, 90,188, 90,189, 90,190, 90,191,90,192, 90,193, 90,194, 90,195, 90,196, 90,197, 90,198, 90,199, 90,200,90,201, 90,202, 90,203, 90,204, 90,205, 90,206, 90,207, 90,208, 90,209,90,210, 90,211, 90,212, 90,213, 90,214, 90,215, 90,216, 90,217, 90,218,90,219, 90,220, 90,221, 90,222, 90,223, 90,224, 90,225, 90,226, 90,227,90,228, 90,229, 90,230, 90,231, 90,232, 90,233, 90,234, 90,235, 90,236,90,237, 90,238, 90,239, 90,240, 90,241, 90,242, 90,243, 90,244, 90,245,90,246, 90,247, 90,248, 90,249, 90,250, 90,251, 90,252, 90,253, 90,254,90,255, 90,256, 90,257, 90,290, 90,259, 90,260, 90,261, 90,262, 90,263,90,264, 90,265, 90,266, 90,290, 90,268, 90,269, 90,270, 90,271, 90,272,90,273, 90,290, 90,290, 90,276, 90,277, 90,278, 90,279, 90,280, 90,281,90,282, 90,283, 90,290, 90,285, 90,286, 90,287, 90,288, 90,289, 90,290,90,291, 90,292, 90,293, 90,294, 90,295, 90,296, 90,297, 90,298, 90,299,90,300, 90,301, 90,302, 90,303, 90,304, 90,305, 90,306, 90,307, 90,308,90,309, 90,310, 90,311, 90,312, 90,313, 90,314, 90,315, 90,316, 90,317,90,318, 90,319, 90,320, 90,321, 90,322, 90,323, 90,324, 90,325, 90,326,90,327, 90,328, 90,329, 90,330, 90,331, 90,332, 90,333, 90,334, 90,335,90,336, 90,337, 90,338, 90,339, 90,340, 90,341, 90,342, 90,343, 90,344,90,345, 90,346, 90,347, 90,348, 90,349, 90,350, 90,351, 90,352, 90,353,90,354, 90,355, 90,356, 90,357, 90,390, 90,359, 90,360, 90,361, 90,362,90,363, 90,364, 90,365, 90,366, 90,390, 90,368, 90,369, 90,370, 90,371,90,372, 90,373, 90,390, 90,390, 90,376, 90,377, 90,378, 90,379, 90,380,90,381, 90,382, 90,383, 90,390, 90,385, 90,386, 90,387, 90,388, 90,389,90,390, 90,391, 90,392, 90,393, 90,394, 90,395, 90,396, 90,397, 90,398,90,399, 90,400, 90,401, 90,402, 90,403, 90,404, 90,405, 90,406, 90,407,90,408, 90,409, 90,410, 90,411, 90,412, 90,413, 90,414, 90,415, 90,416,90,417, 90,418, 90,419, 90,420, 90,421, 90,422, 90,423, 90,424, 90,425,90,426, 90,427, 90,428, 90,429, 90,430, 90,431, 90,432, 90,433, 90,434,90,435, 90,436, 90,437, 90,438, 90,439, 90,440, 90,441, 90,442, 90,443,90,444, 90,445, 90,446, 90,447, 90,448, 90,449, 90,450, 90,451, 90,452,90,453, 90,454, 90,455, 90,456, 90,457, 90,490, 90,459, 90,460, 90,461,90,462, 90,463, 90,464, 90,465, 90,466, 90,490, 90,468, 90,469, 90,470,90,471, 90,472, 90,473, 90,490, 90,490, 90,476, 90,477, 90,478, 90,479,90,480, 90,481, 90,482, 90,483, 90,490, 90,485, 90,486, 90,487, 90,488,90,489, 90,490, 90,491, 90,492, 90,493, 90,494, 90,495, 90,496, 90,497,90,498, 90,499, 90,500, 90,501, 90,502, 90,503, 90,504, 90,505, 90,506,90,507, 90,508, 90,509, 90,510, 90,511, 90,512, 90,513, 90,514, 90,515,90,516, 90,517, 90,518, 90,519, 90,520, 90,521, 90,522, 90,523, 90,524,90,525, 90,526, 90,527, 90,528, 90,529, 90,530, 90,531, 90,532, 90,533,90,534, 90,535, 90,536, 90,537, 90,538, 90,539, 90,540, 90,541, 90,542,90,543, 90,544, 90,545, 90,546, 90,547, 90,548, 90,549, 90,550, 90,551,90,552, 90,553, 90,554, 90,555, 90,556, 90,557, 90,590, 90,559, 90,560,90,561, 90,562, 90,563, 90,564, 90,565, 90,566, 90,590, 90,568, 90,569,90,570, 90,571, 90,572, 90,573, 90,590, 90,590, 90,576, 90,577, 90,578,90,579, 90,580, 90,581, 90,582, 90,583, 90,590, 90,585, 90,586, 90,587,90,588, 90,589, 90,590, 90,591, 90,592, 90,593, 90,594, 90,595, 90,596,90,597, 90,598, 90,599, 90,600, 90,601, 90,602, 90,603, 90,604, 90,605,90,606, 90,607, 90,608, 90,609, 90,610, 90,611, 90,612, 90,613, 90,614,90,615, 90,616, 90,617, 90,618, 90,619, 90,620, 90,621, 90,622, 90,623,90,624, 90,625, 90,626, 90,627, 90,628, 90,629, 90,630, 90,631, 90,632,90,633, 90,634, 90,635, 90,636, 90,637, 90,638, 90,639, 90,640, 90,641,90,642, 90,643, 90,644, 90,645, 90,646, 90,647, 90,648, 90,649, 90,650,90,651, 90,652, 90,653, 90,654, 90,655, 90,656, 90,657, 90,690, 90,659,90,660, 90,661, 90,662, 90,663, 90,664, 90,665, 90,666, 90,690, 90,668,90,669, 90,670, 90,671, 90,672, 90,673, 90,690, 90,690, 90,676, 90,677,90,678, 90,679, 90,680, 90,681, 90,682, 90,683, 90,690, 90,685, 90,686,90,687, 90,688, 90,689, 90,690, 90,691, 90,692, 90,693, 90,694, 90,695,90,696, 90,697, 90,698, 90,699, 90,700, 90,701, 90,702, 90,703, 90,704,90,705, 90,706, 90,707, 90,708, 90,709, 90,710, 90,711, 90,712, 90,713,90,714, 90,715, 90,716, 90,717, 90,718, 90,719, 90,720, 90,721, 90,722,90,723, 90,724, 90,725, 90,726, 90,727, 90,728, 90,729, 90,730, 90,731,90,732, 90,733, 90,734, 90,735, 90,736, 90,737, 90,738, 90,739, 90,740,90,741, 90,742, 90,743, 90,744, 90,745, 90,746, 90,747, 90,748, 90,749,90,750, 90,751, 90,752, 90,753, 90,754, 90,755, 90,756, 90,757, 90,790,90,759, 90,760, 90,761, 90,762, 90,763, 90,764, 90,765, 90,766, 90,790,90,768, 90,769, 90,770, 90,771, 90,772, 90,773, 90,790, 90,790, 90,776,90,777, 90,778, 90,779, 90,780, 90,781, 90,782, 90,783, 90,790, 90,785,90,786, 90,787, 90,788, 90,789, 90,790, 90,791, 90,792, 90,793, 90,794,90,795, 90,796, 90,797, 90,798, 90,799, 90,800, 90,801, 90,802, 90,803,90,804, 90,805, 90,806, 90,807, 90,808, 90,809, 90,810, 90,811, 90,812,90,813, 90,814, 90,815, 90,816, 90,817, 90,818, 90,819, 90,820, 90,821,90,822, 90,823, 90,824, 90,825, 90,826, 90,827, 90,828, 90,829, 90,830,90,831, 90,832, 90,833, 90,834, 90,835, 90,836, 90,837, 90,838, 90,839,90,840, 90,841, 90,842, 90,843, 90,844, 90,845, 90,846, 90,847, 90,848,90,849, 90,850, 90,851, 90,852, 90,853, 90,854, 90,855, 90,856, 90,857,90,890, 90,859, 90,860, 90,861, 90,862, 90,863, 90,864, 90,865, 90,866,90,890, 90,868, 90,869, 90,870, 90,871, 90,872, 90,873, 90,890, 90,890,90,876, 90,877, 90,878, 90,879, 90,880, 90,881, 90,882, 90,883, 90,890,90,885, 90,886, 90,887, 90,888, 90,889, 90,890, 90,891, 90,892, 90,893,90,894, 90,895, 90,896, 90,897, 90,898, 90,899, 90,900, 90,901, 90,902,90,903, 90,904, 90,905, 90,906, 90,907, 90,908, 90,909, 90,910, 90,911,90,912, 90,913, 90,914, 90,915, 90,916, 90,917, 90,918, 90,919, 90,920,90,921, 90,922, 90,923, 90,924, 90,925, 90,926, 90,927, 90,928, 90,929,90,930, 90,931, 90,932, 90,933, 90,934, 90,935, 90,936, 90,937, 90,938,90,939, 90,940, 90,941, 90,942, 90,943, 90,944, 90,945, 90,946, 90,947,90,948, 90,949, 90,950, 90,951, 90,952, 90,953, 90,954, 90,955, 90,956,90,957, 90,990, 90,959, 90,960, 90,961, 90,962, 90,963, 90,964, 90,965,90,966, 90,990, 90,968, 90,969, 90,970, 90,971, 90,972, 90,973, 90,990,90,990, 90,976, 90,977, 90,978, 90,979, 90,980, 90,981, 90,982, 90,983,90,990, 90,985, 90,986, 90,987, 90,988, 90,989, 90,990, 90,991, 90,992,90,993, 90,994, 90,995, 90,996, 90,997, 90,998, 90,999 or 91,000; and96,000 to 97,000 include 96,001, 96,002, 96,003, 96,004, 96,005, 96,006,96,007, 96,008, 96,009, 96,010, 96,011, 96,012, 96,013, 96,014, 96,015,96,016, 96,017, 96,018, 96,019, 96,020, 96,021, 96,022, 96,023, 96,024,96,025, 96,026, 96,027, 96,028, 96,029, 96,030, 96,031, 96,032, 96,033,96,034, 96,035, 96,036, 96,037, 96,038, 96,039, 96,040, 96,041, 96,042,96,043, 96,044, 96,045, 96,046, 96,047, 96,048, 96,049, 96,050, 96,051,96,052, 96,053, 96,054, 96,055, 96,056, 96,057, 96,096, 96,059, 96,060,96,061, 96,062, 96,063, 96,064, 96,065, 96,066, 96,096, 96,068, 96,069,96,070, 96,071, 96,072, 96,073, 96,096, 96,096, 96,076, 96,077, 96,078,96,079, 96,080, 96,081, 96,082, 96,083, 96,096, 96,085, 96,086, 96,087,96,088, 96,089, 96,096, 96,091, 96,092, 96,093, 96,094, 96,095, 96,096,96,097, 96,098, 96,099, 96,100, 96,101, 96,102, 96,103, 96,104, 96,105,96,106, 96,107, 96,110, 96,111, 96,112, 96,113, 96,114, 96,115, 96,116,96,117, 96,118, 96,119, 96,120, 96,121, 96,122, 96,123, 96,124, 96,125,96,126, 96,127, 96,128, 96,129, 96,130, 96,131, 96,132, 96,133, 96,134,96,135, 96,136, 96,137, 96,138, 96,139, 96,140, 96,141, 96,142, 96,143,96,144, 96,145, 96,146, 96,147, 96,148, 96,149, 96,150, 96,151, 96,152,96,153, 96,154, 96,155, 96,156, 96,157, 96,196, 96,159, 96,160, 96,161,96,162, 96,163, 96,164, 96,165, 96,166, 96,196, 96,168, 96,169, 96,170,96,171, 96,172, 96,173, 96,196, 96,196, 96,176, 96,177, 96,178, 96,179,96,180, 96,181, 96,182, 96,183, 96,196, 96,185, 96,186, 96,187, 96,188,96,189, 96,196, 96,191, 96,192, 96,193, 96,194, 96,195, 96,196, 96,197,96,198, 96,199, 96,200, 96,201, 96,202, 96,203, 96,204, 96,205, 96,206,96,207, 96,208, 96,209, 96,210, 96,211, 96,212, 96,213, 96,214, 96,215,96,216, 96,217, 96,218, 96,219, 96,220, 96,221, 96,222, 96,223, 96,224,96,225, 96,226, 96,227, 96,228, 96,229, 96,230, 96,231, 96,232, 96,233,96,234, 96,235, 96,236, 96,237, 96,238, 96,239, 96,240, 96,241, 96,242,96,243, 96,244, 96,245, 96,246, 96,247, 96,248, 96,249, 96,250, 96,251,96,252, 96,253, 96,254, 96,255, 96,256, 96,257, 96,296, 96,259, 96,260,96,261, 96,262, 96,263, 96,264, 96,265, 96,266, 96,296, 96,268, 96,269,96,270, 96,271, 96,272, 96,273, 96,296, 96,296, 96,276, 96,277, 96,278,96,279, 96,280, 96,281, 96,282, 96,283, 96,296, 96,285, 96,286, 96,287,96,288, 96,289, 96,296, 96,291, 96,292, 96,293, 96,294, 96,295, 96,296,96,297, 96,298, 96,299, 96,300, 96,301, 96,302, 96,303, 96,304, 96,305,96,306, 96,307, 96,308, 96,309, 96,310, 96,311, 96,312, 96,313, 96,314,96,315, 96,316, 96,317, 96,318, 96,319, 96,320, 96,321, 96,322, 96,323,96,324, 96,325, 96,326, 96,327, 96,328, 96,329, 96,330, 96,331, 96,332,96,333, 96,334, 96,335, 96,336, 96,337, 96,338, 96,339, 96,340, 96,341,96,342, 96,343, 96,344, 96,345, 96,346, 96,347, 96,348, 96,349, 96,350,96,351, 96,352, 96,353, 96,354, 96,355, 96,356, 96,357, 96,396, 96,359,96,360, 96,361, 96,362, 96,363, 96,364, 96,365, 96,366, 96,396, 96,368,96,369, 96,370, 96,371, 96,372, 96,373, 96,396, 96,396, 96,376, 96,377,96,378, 96,379, 96,380, 96,381, 96,382, 96,383, 96,396, 96,385, 96,386,96,387, 96,388, 96,389, 96,396, 96,391, 96,392, 96,393, 96,394, 96,395,96,396, 96,397, 96,398, 96,399, 96,400, 96,401, 96,402, 96,403, 96,404,96,405, 96,406, 96,407, 96,408, 96,409, 96,410, 96,411, 96,412, 96,413,96,414, 96,415, 96,416, 96,417, 96,418, 96,419, 96,420, 96,421, 96,422,96,423, 96,424, 96,425, 96,426, 96,427, 96,428, 96,429, 96,430, 96,431,96,432, 96,433, 96,434, 96,435, 96,436, 96,437, 96,438, 96,439, 96,440,96,441, 96,442, 96,443, 96,444, 96,445, 96,446, 96,447, 96,448, 96,449,96,450, 96,451, 96,452, 96,453, 96,454, 96,455, 96,456, 96,457, 96,496,96,459, 96,460, 96,461, 96,462, 96,463, 96,464, 96,465, 96,466, 96,496,96,468, 96,469, 96,470, 96,471, 96,472, 96,473, 96,496, 96,496, 96,476,96,477, 96,478, 96,479, 96,480, 96,481, 96,482, 96,483, 96,496, 96,485,96,486, 96,487, 96,488, 96,489, 96,496, 96,491, 96,492, 96,493, 96,494,96,495, 96,496, 96,497, 96,498, 96,499, 96,500, 96,501, 96,502, 96,503,96,504, 96,505, 96,506, 96,507, 96,508, 96,509, 96,510, 96,511, 96,512,96,513, 96,514, 96,515, 96,516, 96,517, 96,518, 96,519, 96,520, 96,521,96,522, 96,523, 96,524, 96,525, 96,526, 96,527, 96,528, 96,529, 96,530,96,531, 96,532, 96,533, 96,534, 96,535, 96,536, 96,537, 96,538, 96,539,96,540, 96,541, 96,542, 96,543, 96,544, 96,545, 96,546, 96,547, 96,548,96,549, 96,550, 96,551, 96,552, 96,553, 96,554, 96,555, 96,556, 96,557,96,596, 96,559, 96,560, 96,561, 96,562, 96,563, 96,564, 96,565, 96,566,96,596, 96,568, 96,569, 96,570, 96,571, 96,572, 96,573, 96,596, 96,596,96,576, 96,577, 96,578, 96,579, 96,580, 96,581, 96,582, 96,583, 96,596,96,585, 96,586, 96,587, 96,588, 96,589, 96,596, 96,591, 96,592, 96,593,96,594, 96,595, 96,596, 96,597, 96,598, 96,599, 96,600, 96,601, 96,602,96,603, 96,604, 96,605, 96,606, 96,607, 96,608, 96,609, 96,610, 96,611,96,612, 96,613, 96,614, 96,615, 96,616, 96,617, 96,618, 96,619, 96,620,96,621, 96,622, 96,623, 96,624, 96,625, 96,626, 96,627, 96,628, 96,629,96,630, 96,631, 96,632, 96,633, 96,634, 96,635, 96,636, 96,637, 96,638,96,639, 96,640, 96,641, 96,642, 96,643, 96,644, 96,645, 96,646, 96,647,96,648, 96,649, 96,650, 96,651, 96,652, 96,653, 96,654, 96,655, 96,656,96,657, 96,696, 96,659, 96,660, 96,661, 96,662, 96,663, 96,664, 96,665,96,666, 96,696, 96,668, 96,669, 96,670, 96,671, 96,672, 96,673, 96,696,96,696, 96,676, 96,677, 96,678, 96,679, 96,680, 96,681, 96,682, 96,683,96,696, 96,685, 96,686, 96,687, 96,688, 96,689, 96,696, 96,691, 96,692,96,693, 96,694, 96,695, 96,696, 96,697, 96,698, 96,699, 96,700, 96,701,96,702, 96,703, 96,704, 96,705, 96,706, 96,707, 96,708, 96,709, 96,710,96,711, 96,712, 96,713, 96,714, 96,715, 96,716, 96,717, 96,718, 96,719,96,720, 96,721, 96,722, 96,723, 96,724, 96,725, 96,726, 96,727, 96,728,96,729, 96,730, 96,731, 96,732, 96,733, 96,734, 96,735, 96,736, 96,737,96,738, 96,739, 96,740, 96,741, 96,742, 96,743, 96,744, 96,745, 96,746,96,747, 96,748, 96,749, 96,750, 96,751, 96,752, 96,753, 96,754, 96,755,96,756, 96,757, 96,796, 96,759, 96,760, 96,761, 96,762, 96,763, 96,764,96,765, 96,766, 96,796, 96,768, 96,769, 96,770, 96,771, 96,772, 96,773,96,796, 96,796, 96,776, 96,777, 96,778, 96,779, 96,780, 96,781, 96,782,96,783, 96,796, 96,785, 96,786, 96,787, 96,788, 96,789, 96,796, 96,791,96,792, 96,793, 96,794, 96,795, 96,796, 96,797, 96,798, 96,799, 96,800,96,801, 96,802, 96,803, 96,804, 96,805, 96,806, 96,807, 96,808, 96,809,96,810, 96,811, 96,812, 96,813, 96,814, 96,815, 96,816, 96,817, 96,818,96,819, 96,820, 96,821, 96,822, 96,823, 96,824, 96,825, 96,826, 96,827,96,828, 96,829, 96,830, 96,831, 96,832, 96,833, 96,834, 96,835, 96,836,96,837, 96,838, 96,839, 96,840, 96,841, 96,842, 96,843, 96,844, 96,845,96,846, 96,847, 96,848, 96,849, 96,850, 96,851, 96,852, 96,853, 96,854,96,855, 96,856, 96,857, 96,896, 96,859, 96,860, 96,861, 96,862, 96,863,96,864, 96,865, 96,866, 96,896, 96,868, 96,869, 96,870, 96,871, 96,872,96,873, 96,896, 96,896, 96,876, 96,877, 96,878, 96,879, 96,880, 96,881,96,882, 96,883, 96,896, 96,885, 96,886, 96,887, 96,888, 96,889, 96,896,96,891, 96,892, 96,893, 96,894, 96,895, 96,896, 96,897, 96,898, 96,899,96,900, 96,901, 96,902, 96,903, 96,904, 96,905, 96,906, 96,907, 96,908,96,909, 96,910, 96,911, 96,912, 96,913, 96,914, 96,915, 96,916, 96,917,96,918, 96,919, 96,920, 96,921, 96,922, 96,923, 96,924, 96,925, 96,926,96,927, 96,928, 96,929, 96,930, 96,931, 96,932, 96,933, 96,934, 96,935,96,936, 96,937, 96,938, 96,939, 96,940, 96,941, 96,942, 96,943, 96,944,96,945, 96,946, 96,947, 96,948, 96,949, 96,950, 96,951, 96,952, 96,953,96,954, 96,955, 96,956, 96,957, 96,996, 96,959, 96,960, 96,961, 96,962,96,963, 96,964, 96,965, 96,966, 96,996, 96,968, 96,969, 96,970, 96,971,96,972, 96,973, 96,996, 96,996, 96,976, 96,977, 96,978, 96,979, 96,980,96,981, 96,982, 96,983, 96,996, 96,985, 96,986, 96,987, 96,988, 96,989,96,996, 96,991, 96,992, 96,993, 96,994, 96,995, 96,996, 96,997, 96,998,96,999 or 97,000.

Examples of insertion sites are provided in Table 2. As indicated above,the sites are based on GenBank Accession No. AJ004801 or its equivalent.

The present disclosure teaches BoHV-1 vaccine vector comprising a BoHV-1genome derived from BoHV-1 strain V155 having heterologous geneticmaterial encoding at least one antigen from a bovine pathogen insertedinto a site on the BoHV-1 genome selected from nucleotides 16600 to16700, 22400 to 22500; 40,700 to 40,800; 58,000 to 59,000; 67,000 to68,000; 74,000 to 76,000; 84,000 to 85,000; 90,000 to 91,000; and 96,000to 97,000 of BoHV-1 reference sequence GenBank Accession No. AJ004801 orat a functionally equivalent site in another BoHV-1. Examples includethe sites listed in Table 2.

There are a range of other sites into which the heterologous geneticmaterial can be inserted. All such sites are enabled herein.

Another aspect taught herein is a method of producing a vaccine againstat least one antigen from a bovine pathogen, the method comprising:

(i) incorporating a BoHV-1 genome from a low virulence BoHV-1 into abacterial artificial chromosome (BAC) vector to form a BoHV-1 pre-vectorBAC construct;

(ii) inserting genetic material encoding the at least one antigen intothe BoHV-1 pre-vector BAC construct via an inducible recombinationsystem to generate a recombinant BoHV-1-BAC (rBoHV-1-BAC) vector;

(iii) transforming and amplifying the rBoHV-1-BAC vector in a bacterialhost; and

(iv) purifying and isolating the rBoHV-1-BAC vector from the bacterialhost and formulating the vector into a vaccine composition.

The present disclosure teaches a vaccination protocol in bovine animalssuch as feedlot cattle, diary cattle and other closely housed cattle.The vaccine preparation may be administered by a range of local andsystemic protocols such as intra-nasal, oral, intra-muscular,sub-lingual, intravenous, subcutaneous or intra-arterial injection, skinspray or other convenient route including intra-vaginal and intra-rectaladministration. An intra-nasal route is particularly efficacious. Theformulation may be a standard pharmaceutical preparation. In anembodiment, the formulation is freeze-dried and re-constituted prior touse.

Hence, a vaccine enabled herein is generally prepared as or is suitablefor re-constitution as an injectable or nasal-adminsitratable liquidsolution or suspension or freeze-dried preparation. The vaccine may alsobe emulsified. Prior to use, a pharmaceutically acceptable diluent,carrier or excipient. The vaccine formulation may also contain auxiliarysubstances such as wetting or emulsifying agents, pH buffering agentsand/or adjuvants.

Accordingly, taught herein is a vaccine formulation comprising a BoHV-1genome from a low virulence BoHV-1 having genetic material encoding theat least one antigen which is heterologous to BoHV-1 inserted betweentwo converging BoHV-1 genes wherein the insertion does not substantiallydown-regulate expression of the BoHV-1 genes; the formulation being infreeze-dried form or further comprising one or more pharmaceuticallyacceptable carriers, diluents and/or excipients.

The formulation may further comprise a BoHV-1 genome from a lowvirulence BoHV-1 which when expressed produces an antigen to which animmune response is capable of being generated, the BoHV-1 genome furthercomprising genetic material encoding at least one other antigenheterologous to BoHV-1 inserted between two converging BoHV-1 geneswherein the insertion does not substantially down-regulate expression ofthe BoHV-1 genes and wherein the heterologous antigen induces an immuneresponse; the formulation being in freeze-dried form or furthercomprising one or more pharmaceutically acceptable carriers, diluentsand/or excipients.

The present disclosure further enables a diagnostic assay toserologically distinguish between vaccinated and non-vaccinated bovineanimals. Generally, a standard antibody assay is conducted to detectantibodies expected to have arisen following vaccination with the BoHV-1recombinant vaccine. In an embodiment, one of the heterologous antigensexpressed by the BoHV-1 vector is a marker protein such as a greenfluorescent protein. This is a convenient marker for the effectivenessof vaccination and as a proprietary tag.

Enabled herein is a business method for managing bovine animals in aconfined location, the business method comprising vaccinating a bovineanimal against at least one antigen from a bovine pathogen, the methodcomprising administering to the bovine animal, a humoralimmunity-inducing or cell-mediated immunity-inducing effective amount ofa BoHV-1 genome from a low virulence BoHV-1 having genetic materialencoding the at least one antigen which is heterologous to BoHV-1inserted between two converging BoHV-1 genes wherein the insertion doesnot substantially down-regulate expression of the BoHV-1 genes to reducethe incidence of spread of BRDC thereby maintaining economic viabilityof the bovine animals.

The business method incorporates a management protocol for maintainingbovine animals and may include a fee for service for practitioners totest for BRDC, vaccinate the bovine animals and then maintainserological analysis of the herd of animals.

The cost of performing the business method may be met by the owner ofthe herd of bovine animals or passed onto consumers.

Serological testing also enables epidemiological studies to beconducted.

Hence, a vaccine is provided herein against at least one antigen from abovine pathogen, the vaccine comprising a bovine herpes virus-1 (BoHV-1)genome from a low virulence BoHV-1 having genetic material encoding theat least one antigen which is heterologous to BoHV-1 inserted betweentwo converging BoHV-1 genes wherein the insertion does not substantiallydown-regulate expression of the BoHV-1 genes. In an embodiment, thegenetic material encoding the at least one antigen is inserted into theBoHV-1 genome via an inducible recombination system, such as via GETrecombination.

The genetic material encoding the at least one antigen is convenientlyinserted between the polyadenylation signals of two converging genes ata site selected from between 16600 to 16700, 22400 to 22500; 40,700 to40,800; 58,000 to 59,000; 67,000 to 68,000; 74,000 to 76,000; 84,000 to85,000; 90,000 to 91,000; and 96,000 to 97,000 of BoHV-1 referencesequence GenBank Accession No. AJ004801 or at a functionally equivalentsite in another BoHV-1. In an embodiment, the at least one antigen isinserted between two converging genes at a site selected from a sitelisted in Table 2. In another embodiment, the at least one antigen isinserted between two converging genes at a site selected from between16602 to 16603 and 22421 to 22470.

Conveniently, the vaccine provides a BoHV-1 which produces a BoHV-1antigen and/or an antigen selected from the list consisting of anantigen from bovine viral diarrhoea virus (BVDV) and an antigen from amicroorganism.

Examples of BVDV antigens are glycoprotein E0 and glycoprotein E2.Examples of microorganisms as a source of antigens include Mycoplasmabovis, a Salmonella species, Pateurella multocida, Manhiemia haemolyticaand Haemophilus somnus.

In an embodiment, the low virulence BoHV-1 strain is strain V155. Thevaccine enabled herein may further comprising additional geneticmaterial encoding another antigen inserted via restriction endonucleasedigestion. The vaccine may also be formulated in a pharmaceuticalcomposition such as a pharmaceutical composition suitable for nasaladministration.

A method for vaccinating a bovine animal against at least one antigenfrom a bovine pathogen is contemplated herein by administering to thebovine animal, a humoral immunity-inducing or cell-mediatedimmunity-inducing effective amount of a bovine herpes virus-1 (BoHV-1)genome from a low virulence BoHV-1 having genetic material encoding theat least one antigen which is heterologous to BoHV-1 inserted betweentwo converging BoHV-1 genes wherein the insertion does not substantiallydown-regulate expression of the BoHV-1 genes. The genetic materialencoding the at least one antigen may be inserted into the BoHV-1 genomevia an inducible recombination system such as by GET recombination.

As indicated above, the genetic material encoding the at least oneantigen is inserted between the polyadenylation signals of twoconverging genes at a site selected from between 16600 to 16700, 22400to 22500; 40,700 to 40,800; 58,000 to 59,000; 67,000 to 68,000; 74,000to 76,000; 84,000 to 85,000; 90,000 to 91,000; and 96,000 to 97,000 ofBoHV-1 reference sequence GenBank Accession No. AJ004801 or at afunctionally equivalent site in another BoHV-1 including between twoconverging genes at a site selected from between 16600 to 16612 and22400 to 22493 including between two converging genes at a site selectedfrom between 16602 to 16603 and 22421 to 22470 including between twoconverging genes at a site selected from sites listed in Table 2.

In accordance with this method, the BoHV-1 produces a BoHV-1 antigenand/or at least one antigen is selected from the list consisting of anantigen from bovine viral diarrhoea virus (BVDV) and an antigen from amicroorganism.

Examples of the BVDV antigen is glycoprotein E0 or glycoprotein E2.Examples of a microorganism is Mycoplasma bovis, a Salmonella species,Pateurella multocida, Manhiemia haemolytica or Haemophilus somnus.

As indicated above the low virulence BoHV-1 strain includes V155.Additional genetic material encoding another antigen may also beinserted via restriction endonuclease digestion.

A method of producing a vaccine against at least one antigen from abovine pathogen, is enabled herein by:

(i) incorporating a BoHV-1 genome from a low virulence BoHV-1 into abacterial artificial chromosome (BAC) vector to form a BoHV-1 pre-vectorBAC construct;

(ii) inserting genetic material encoding the at least one antigen intothe BoHV-1 pre-vector BAC construct via an inducible recombinationsystem to generate a recombinant BoHV-1-BAC (rBoHV-1-BAC) vector;

(iii) transforming and amplifying the rBoHV-1-BAC vector in a bacterialhost; and

(iv) purifying and isolating the rBoHV-1-BAC vector from the bacterialhost and formulating the vector into a vaccine composition.

The inducible recombination system and sites of insertion are asdisclosed above as are the antigens and their sources.

A cultured cell transfected with the rBoHV-1-BAC vector is also enabledherein.

A method taught therein is the vaccination against bovine respiratorydisease complex (BRDC) in cattle, the method comprising administering tothe cattle a humoral immunity-inducing or cell-mediatedimmunity-inducing effective amount of a bovine herpes virus-1 (BoHV-1)genome from a low virulence BoHV-1 having genetic material encoding theat least one antigen which is heterologous to BoHV-1 inserted betweentwo converging BoHV-1 genes wherein the insertion does not substantiallydown-regulate expression of the BoHV-1 genes.

Further contemplated herein is the use of a bovine herpes virus-1(BoHV-1) genome from a low virulence BoHV-1 having genetic materialencoding the at least one antigen which is heterologous to BoHV-1inserted between two converging BoHV-1 genes wherein the insertion doesnot substantially down-regulate expression of the BoHV-1 genes in themanufacture of a medicament in the vaccination of cattle against abovine pathogen.

Aspects enabled herein are described by the following non-limitingExamples.

Example 1 BoHV-1 Strain

The low virulence strain of BoHV-1 used was strain V155, originallydescribed by Snowden (1964) supra. Nucleotide position numbers are basedon GenBank Accession No. AJ004801.

Example 2 Construction of a Recombinant Bovine Herpesvirus-1 (BoHV-1)

The BoHV-1 strain V155 was propagated in CRIB-1 cells (ATCC numberCRL-11883), a pestivirus resistant derivative of MDBK cells. The CRIB-1cells were maintained in Hank's minimal essential medium (H-MEM)containing antibiotics/antimyotics, non-essential amino acids, glutaMAX,25 mM Hepes and 5% v/v donor calf sera at 37° C. All reagents utilizedfor cell and virus propagation were obtained from Invitrogen Australiaunless otherwise stated.

CRIB-1 cells into six well plates (Corning) at 5×10⁵ cells/well 24 hprior to transfection and incubate at 37° C. in an atmosphere of 5% v/vCO₂. For each transfection, diluted 1-2 μg of DNA to 100 μl usingOptiMEM and mixed with 8 μl of Lipofectamine diluted to 100 μl usingOptiMEM. The resultant mixture was incubated the mixture at roomtemperature for 45 min for formation of lipid/DNA complexes. Thereaction volume was increased to 1 ml using OptiMEM and add to the cellmonolayers which have been washed twice with OptiMEM. The transfectedmonolayers were then incubate the transfected monolayers at 37° C. with5% v/v CO₂ for 16-18 h prior to the addition of 1 ml OptiMEM containing10% v/v donor calf sera. 24 h the remove the transfection liquid andreplaced with maintenance media. The monolayers for the development ofCPE for up to 7 days post-transfection.

In order to purify the BoHV-1 genomic DNA the MDBK variant CRIB-1 cellswere infected with BoHV-1 strain V155 at an MOI of 5 and the infectionallowed to proceed to completion. The cell culture supernatant was thenclarified by centrifugation at 5000 g for 10 min Mature BoHV-1 virionswere pelleted by centrifugation at 120000 g for 2 h. The BoHV-1 genomicDNA was recovered from the pelleted virus using the Qiagen genomic DNAextraction kit essentially as described by the manufacturer. The viralpellets are resuspend in Genomic DNA extraction buffer at a ratio of1:65 of starting supernatant volume. Following elution from the columnthe BoHV-1 DNA was stored in aliquots at −20° C. HindIII was used todigest 1-2 μg of the DNA for comparison to known digestion profile ofBoHV-1.

To facilitate the insertion of transgenes into the TK gene of BoHV-1using GET homologous recombination, the deletion/insertion vector, pTKdel was constructed. This vector contains two segments of the BoHV-1thymidine kinase gene, TKleft and TKright, for use as recombinationarms.

PCR for the BoHV-1 genome was carried out using Taq polymerase buffer(10 mM Tris-HCl, 1.5 mM MgCl₂, 50 mM KCl, pH8.5); 1.25 mM each of dATP,dCTP, dGTP and dTTP; 12.5 μM of each primer; 1 U of Taq DNA polymerase;10-20 ng of genomic DNA 5% v/v DMSO; 10% v/v glycerol. These componentshad a final reaction volume of 20 μl.

The PCR cycling conditions used were; denaturation at 94° C. for 4 min;35 cycles of 94° C. for 20 sec, 60° C. for 20 sec and 72° C. 120 sec;followed by 72° C. for 10 min and subsequently held at 4° C. Cycling wasperformed in a Hybaid Sprint thermocycler. After cycling the PCR productwas resolved on 1% w/v agarose gel, the product excised and the DNArecovered using a Qiagen gel extraction kit according to themanufacturer's instructions.

TKleft and TKright were amplified from purified BoHV-1 genomic DNA byPCR. After amplification the products were purified using a Qiagen PCRpurification column according to the manufacturer's instructions. TheTKleft PCR product was digested with KpnI and SalI, gel purified andligated into pBluescript-SK+ (Stratagene) which had also been digestedwith KpnI/SalI. The presence of the TKleft product was confirmed bysequencing. The TKright PCR product was cloned into the plasmidcontaining the TKleft product following EcoRI and SpeI digestion usingstandard cloning procedures. The resultant plasmid was called pTKdel(see Mahoney et al. (2002) supra). The primers used for the PCRamplification of the TK targeting regions are shown in Table 3. NsiIsites were incorporated into the Tkleft5′ and Tkright3′ primers to allowthe excision of the transgene product from the pTKdel vector forrecombination experiments. Four unique restriction endonuclease sitesare present between the two TK crossover regions to allow the insertionof transgenes for transfer to the BoHV-1 genome.

In order to transfer the bacterial artificial chromosome (BAC) to thegenome of BoHV-1, the BAC vector pBello-BAC II was digested with HindIIIand gel purified. The digested BAC vector was ligated into pTKdel whichhad been digested with HindIII and dephosphorylated. Followingtransformation into E. coli strain XL1-Blue cells transformants wereplated on selective agar containing 12.5 μg/ml chloramphenicol (CAP) and100 μg/ml ampicillin. Insertion of the BAC vector was confirmed byexcision with HindIII from DNA recovered from the resultant colonies.The TK deletion fragment (TK-BAC), containing the BAC vector flanked bythe TKleft and TKright, was excised from pTKdel-BAC by digestion withNsiI and gel purified.

To promote homologous recombination between the BAC-TK fragment andBoHV-1 genomic DNA, purified BoHV-1 DNA was digested with NsiI anddephosphorylated with bacterial alkaline phosphatase (Pharmacia). TheBAC-TK fragment and NsiI digested BoHV-1 genomic DNA were co-transfectedinto CRIB-1 cells as described above. After 18-24 h the transfectionmixture was removed and replaced with complete H-MEM containing 2 mMN,N′-hexamethylene-bis-acetamide (ICN) to promote viral genetranscription. The resultant viral supernatants were passaged once inCRIB-1 cells. The insertion of the BAC vector into the BoHV-1 genome wasconfirmed by a PCR assay specific for the chloramphenicol resistancegene using the primer pair ChloramF and ChloramR.

PCR templates were prepared by incubation of 10 μl of viral supernatantwith 10 μl of lysis buffer (10 mM Tris-HCl pH8.0 containing 0.45% v/vTriton X-100 and 0.45% v/v Tween 20) with 2 μl of 10 mg/ml proteinase Kfollowed by incubation at 60° C. for 2 h. The proteinase K wasinactivated at 95° C. for 15 min. PCR reactions were performed using 1to 2 μl of this preparation as template. Following PCR detection of theCAP resistance gene within the BoHV-1 genome bulk genomic DNA wasrecovered from virus particles as described above. To facilitatetransformation and growth in a bacterial host the purified BoHV-1genomic DNA was circularized using standard ligation procedures.Aliquots of the ligation mixtures were electroporated into E. coli DH10Bcells (1.5 kV, 100 Ω, 25 μF, Electroporator II; Invitrogen, San Diego).Following electroporation DH10B cells were recovered in 960 μl of SOBand incubated at 37° C. for 5 to 6 h with gentle shaking. Aliquots ofthe electroporation mix were plated onto LB plates containing 12.5 μg/mlCAP. Colonies were allowed to develop for 24 to 48 h at 37° C.

CAP resistant colonies were inoculated into 5 ml of LB broth containing12.5 μg/ml CAP and grown at 37° C. for 16 h. BAC DNA was recovered usingthe standard alkaline lysis method. The HindIII digestion profiles ofthese BAC clones were compared to the HindIII profile of BoHV-1 genomicDNA. BAC clones with a similar HindIII profiles to genomic DNA weretransfected into CRIB-1 cells as described above. The transfections weremonitored daily for the development of CPE considered typical of BoHV-1.

Recombination of BoHV-1 BAC in DH10B occurred by co-electroporating theplasmid, pGETrec (available from by the Murdoch Childrens ResearchInstitute, Melbourne, Australia), was electroporated into DH10B cellsharbouring pBACBHV37 (BoHV-1 infectious clone). The DH10B cellscontaining both plasmids were selected on agar containing 12.5 μg/ml CAPand 100 μg/ml ampicillin Electrocompetent cells of double resistantDH10B cells were prepared with 0.2% w/v arabinose induction during thelog phase of cell growth to enable homologous recombination aspreviously described. The PCR amplified transgene of interest is thenelectroporated into these cells. Following recovery from theelectroporation cuvette in SOC broth the cells are allowed to recover at37° C. for at least 6 hr with shaking. All of the recovered cells on tochloramphenicol selective plates along with the appropriate antibioticto select for the transgene.

To demonstrate that GET recombination could be utilized to modify theBoHV-1 genome, two different rBoHV-1 viruses were created. A rBOHV-1 wascreated where:

(i) The gene encoding gE was deleted. This was carried out by using aminimal kanamycin resistance cassette (Kan^(R)) which was amplified byPCR from the transposon EZ::TN<KAN-1> (Epicentre Technologies). The PCRprimers: gE-KanF and gE-KanR, utilized included regions of 50 bphomology to the 5′ (base number 121671-121720 of AJ004801) and 3′ ends(base number 123371-123420 of AJ004801) of the gE gene. The resultantproduct, gE-Kan^(R), was 1300 bp in length and was recovered followingagarose gel separation using a gel extraction kit according to themanufacturer's instructions (Qiagen).

(ii) A heterologous gene encoding green fluorescent protein (GFP) wasinserted into the V155 genome at the Nsil site REMR. This was carriedout by inserting the GFP expression cassette into the genome locatedbetween UL46 (protein virion protein tegument) and UL44 (glycoprotein Cusing pGETrec facilitated recombination). the resultant product wasrecovered following agarose gel separation using a gel extraction kitaccording to the manufacture's instructions (Qiagen).

Other sites are selected from those listed in Table 2.

Approximately 200 ng of the gel purified PCR product was electroporatedinto the electrocompetent DH10B (pBACBHV37, pGETrec) as previouslydescribed. Recombinant colonies were identified by plating on LB platescontaining 12.5 μg/ml CAP and 50 μg/ml kanamycin. PCR and Southern blotanalysis confirmed the replacement of the gE gene with the Kan^(R)cassette.

Viral genomic DNA for Southern blotting was purified as described below.Cell monolayers were infected with BoHV-1 strain V155 at an MOI of 5 andthe infection was allowed to proceed until approximately 40% of thecells showed a “rounding up” type of morphology. Following removal ofthe growth medium the monolayer was gently washed twice with PBS at 0°C. Cell lysis buffer at 0° C. (10 mM sodium phosphate, pH 7.3 containing1% v/v Nonidet P-40) was added to each flask (4 ml per 175 cm²) and theflasks rocked so that the lysis buffer contacted the entire monolayer.The lysis buffer was removed and placed on ice, a further 4 ml added toeach flask with gentle rocking it was removed and added to the initiallysis solution. The lysates were clarified at 4300 g for 10 min at 4° C.The supernatant was collected and centrifuged at 112700 g for 100 min at5° C. The viral pellet was resuspended in 500 μl G2 buffer (Qiagen).Proteinase K (25 μl of 10 mg/ml) was added to the resuspended viralpellet followed by incubation at 50° C. for 1 h. Genomic viral DNA wasrecovered using genomic tip 20/G (Qiagen) as follows. The genomic tip20/G was equilibrated with 1 ml of QBT buffer. The proteinase K treatedmaterial was diluted with an equal volume of QBT buffer and loaded on tothe genomic tip (660 μl per tip). Tips were washed twice with 7.5 ml ofQC buffer. Viral DNA was eluted from the tip with 2×1.5 ml QF buffer at50° C. The DNA was precipitated, washed once with 75% v/v ethanol andresuspended in 50 μl of 10 mM Tris-HCl, pH 8.5.

Restriction enzyme digested DNA samples were electrophoresed on a 1% w/vgel in 0.5×TBE buffer for 13.5 h using field inversion gelelectrophoresis (FIGE) apparatus (Biorad) at 5° C. The switch time rampwas 0.1 to 2 s linear shape with a forward voltage of 180 V and areverse voltage of 120 V. The DNA fragments were transferred to Hybond-Nnon-charged membrane (Amersham) using capillary action and the DNA wasfixed to the membrane using UV light. Probes were labeled withDIG-II-dUTP (Roche Molecular Biosystems). Probes were synthesized by PCRusing a reaction mix containing: Taq polymerase buffer (10 mM Tris-HCl,1.5 mM MgCl₂, 50 mM KCl, pH8.5), 1.25 mM each of dATP, dCTP, dGTP anddTTP, 1.25 μM DIG-II-dUTP, 1 U Taq Polymerase (Roche MolecularBiosystems), and 5 ng template DNA in a final volume of 20 μl. PCRreaction conditions were 94° C. for 3 min, 94° C. for 30 sec, 55° C. for30 sec, 68° C. for 1 min for 35 cycles, 68° C. for 6 min. Following gelpurification PCR probes were hybridized to membranes for 12-16 h at 55°C. (DIG System User's Guide for Filter Hybridisation, Roche MolecularBiosystems). Hybridizations were carried out in rotating bottles in ahybridization oven (Hybaid). Membranes were washed in dishes on ashaking platform at RT.

The replication kinetics of the various rBoHV-1 were determined usingstandard virological techniques. Briefly, 1 TCID₅₀ of virus was allowedto infect 1×10⁵ CRIB-1 cells plated in 24-well plates for 90 min at 37°C. with 5% v/v CO₂ atmosphere. Any extracellular virus was theninactivated by addition of sodium citrate solution (40 mM sodiumcitrate, 10 mM KCl, 135 mM NaCl, pH 3.0), the cell layers were thenwashed twice with PBS and 1 ml of maintenance media added and incubatedat 37° C. in a 5% CO₂ atmosphere. Viral supernatants and cell pelletswere collected at 2, 4, 6, 12, 24, 48 and 72 h PI and frozen at −70° C.until required. The TCID₅₀ of each supernatant from each time point wasthen determined in triplicate. Following one freeze/thaw cycle theTCID₅₀ of intracellular virus was also determined for each time point intriplicate.

TABLE 3 Product (size Sequence bp) and Primer Primer Sequence ID NO.Plasmid TKleft5′ 5′-GT GGTACC ATGCAT CTGATACCCCTTCGCCCGCTACTG-3′  1Tkleft        KpnI   NsiI (301 bp) TKleft3′5′-TTTGC GTCGAC CCACTCCAGCGCGTCCCAG-3′  2 pTKdel           SalITKright5′ 5′-AT GAATTC GCCGCGCTCGCAGACCCCA-3′  3 TKright,        EcoRI(337 bp), TKright3′ 5′-GGACTAGTCATGCATCTCTAGCGCGAACTGACG-3′  4 pTKdel      SpeI   NsiI TK-probe ChloramF 5′-TCACTGGATATACCACCGTTGA-3′  5CAP^(R) gene, ChloramR 5′-TCACCGTAACACGCCACATCTT-3′  6 (402 bp) gE-KanF5′-GGGGAACGGCGCACGCGAGAGGGTTCGAAAAGGGCATTTGGCAA  7 CAP^(R)-probeTGCAAC-ATTTAAAT-ccacgttgtgtctcaaaatctctgatg-3′         SwaI gE-Kan^(R)5′-TCGCGCTGCTACCACGGTGTAATCTGGTGCGGCCGGGGTCCG  8 gE-Kan^(R)CGCTGGCG-ATTTAAAT-cggttgatgagagctttgttgtaggtg-3′ (1237 bp)          SwaI BHV1.3 5′-GGG CAT TTG GCA ATG CAA C-3′  9 gE-probe BHV1.65′-CGT CTC GTA TAT GCG GAT G-3′ 10 (845 bp) Kan^(R)fwd5′-GGT ATT AGA AGA ATA TCC TGA TTC-3′ 11 Kan^(R)-probe Kan^(R)rev5′-CTC ATC GAG CAT CAA ATG AAA CT-3′ 12 (483 bp)

Example 3 Assessment of the Transmissibility of the rBoHV-1

The aim of this trial was to determine if the recombinant (geneticallymodified, GM) virus was capable of transmitting from vaccinated cattleto other cattle located at varying distances from the vaccinates. otherruminants (sheep and goats) were also located at varying positionsrelative to the vaccinated cattle to determine if the GM virus wastransmissible to these ruminant species.

All cattle, sheep and goats were negative to BoHV-1 and Bovine viraldiarrhoea virus (BVDV) specific antibodies prior to the commencement ofthe animal trial. Animals of each species were randomly assigned to thefollowing groups:

Sentinel Group A: Cattle (4), Sheep (4) and Goats (4) [note sheep andgoats were penned together] located approximately 23 meters from theanimal house;

Sentinel Group B: Cattle (2×2), Sheep (4) and Goats (4), located in pensin the Western end of the animal house;

Sentinel Group C: Cattle (2×2), Sheep (4) and Goats (4), located in theEastern end of the animal house in pens opposite the vaccinated cattle;and

Vaccine Contact Group: Vaccinated cattle (4) and Contact cattle (4),located in the Eastern end of the animal house.

Environmental swabs were taken from various locations in and around theanimal house to test for the presence of the GM virus persisting outsideits natural host (cattle) on the same day as animal samples werecollected.

Prior to vaccination (Day 0) blood (20 ml) and nasal swabs werecollected from all animals. The rectal temperature of all animals wasalso recorded. Cattle (94) were then vaccinated intra-nasally with 2 mlof prototype vaccine (BoHV-1 TK-E2+). Vaccinated cattle were penned withan unvaccinated bovine. For 14 days following vaccination (Day 1-14)nasal swabs were collected from all animals and rectal temperaturesrecorded for all animals. The trail concluded on Day 28post-vaccination. At this time blood (20 ml) and nasal swabs werecollected from all animals. In addition, rectal temperatures from allanimals were recorded. After the collection of these samples all animalswere euthanized and tissue samples (heart, lung, kidney, spleen, muscle,liver, brain and trigeminal ganglia) collected. Carcases were disposedof via high temperature incineration.

Example 4 Comparison of the Wet or Freeze-Dried gmBoHV-1 Preparations

The aim of this trial was to compare the efficacy of gmBoHV-1 as a wetpreparation and freeze-dried preparation to Rhinogard (Trade Mark)provided by Q-Vax Pty Ltd.

All cattle were negative to BoHV-1 and BVDV specific antibodies prior tothe commencement of the animal trial. Cattle were randomly assigned tothe following groups:

Group 1: Unvaccinated;

Group 2: Vaccinated intra-nasally with 1-2 ml of vaccine (gmBoHV-1) into1 nostril;

Group 3: Vaccinated intra-nasally with 1-2 ml of vaccine (FD-gmBoHV-1)into 1 nostril;

Group 4: Vaccinated intra-nasally with 1-2 ml of vaccine (Rhinogard)into 1 nostril.

Environmental swabs were taken from various locations in and around theanimal house to test for the presence of the GM virus persisting outsideits natural host (cattle) on each day that animal samples werecollected.

Prior to vaccination (Day 0) blood (20 ml), nasal swabs and nasal tamponswabs were collected from all cattle. The rectal temperatures and weight(combined pair weight) of all cattle was also recorded. Groups 2 and 4were then vaccinated intra-nasally with the appropriate formulation.Freeze-dried gmBoHV-1 was reconstituted immediately prior toinstillation. It was planned to administer the vaccine using acommercial applicator, however, due to problems in getting this to work,the vaccinations were delivered using a syringe as performed in previousresearch.

For 7 days following vaccination (Day 1-7) nasal swabs were collectedfrom all animals and clinical signs were recorded for all animals.

All cattle were challenged with the BoHV-1 strain Q3932 on Day 14post-vaccination as described below. Prior to the challenge nasal swabsand clinical signs were recorded for all animals. The cattle were thenchallenged with an intra-nasal instillation of 10⁷ TCID₅₀ BoHV-1 strainQ3932. After BoHV-1 challenge, nasal swabs were collected from allcattle and clinical assessments made on a daily basis (Day 15 to 18)using the scoring method described in Table 4.

TABLE 4 Parameter Score Clinical Sign Residual feed ration 0  0-25% 125-50% 2 50-75% 3 75-100% Coughing 0 Absent 1 cough due to exercise 2coughing in pen Demeanour 0 normal 1 lethargic 2 cast Nasal discharge(N) 0 no discharge 1 mild sero-mucous discharge 2 moderate sero-mucousdischarge 3 mucous discharge with globules or small strands ofmucopurulent exudate 4 thick mucopurulent exudate 5 thick mucopurulentexudate hanging from nostrils Rectal temperature (° C.) AM PM Weight¹(kg) Other Observations

-   -   Clinical signs and parameters used in the clinical assessment of        animals before and following challenge. Respiration rate was        calculated by multiplying the number of breaths taken over 15        seconds by four. Respiration rate was observed in pens prior to        daily sampling. Recta temperatures were taken in the AM and        repeated later in the day if a significant elevation was        observed. Other clinical notes were also recorded as required,        for example, audible breathing. clinical signs were assigned a        numerical score (S). Weight¹ was recorded as a combined measure        for the cattle as the animals were more settled for sampling        procedures when handled in the crush as pairs.

All cattle were challenged with M. haemolytica on Day 18post-vaccination. Prior to the challenge, nasal swabs were collected andclinical signs were recorded for all animals. The cattle were thenchallenged with the intranasal instillation of approximately 5×10⁹ cfuof M. haemolytica. After this secondary challenge, nasal swabs werecollected from all cattle and clinical assessments made on a daily basis(Day 19 to 26) using the scoring method.

The trial concluded on Day 35 post-vaccination. At this time blood (20ml), nasal swabs and nasal tampon swabs were collected from each animal.In addition, rectal temperatures from all animals were recorded. Afterthe collection of these samples all animals were euthanized and tissuesamples (heart, lung, kidney, spleen, muscle, liver, brain andtrigeminal ganglia) were collected. Carcases were disposed of via deepburial.

Example 5 Effects of Pre-Existing Immunity on Vaccine Efficacy

The aim of this trial was to determine if pre-existing immunity toeither BoHV-1 or BVDV would affect the efficacy of the gmBoHV-1 vaccine.For example, if an animals was antibody positive for BoHV-1 would thegmBoHV-1 still be able to induce BVDV immunity (see Table 5).

TABLE 5 Assessment of the effect of pre-existing immunity on theefficacy of the recombinant BoHV-1 vaccine Status at Number of TreatmentChallange vaccination¹ Animals Vaccinated BoHV-1/Mh² BoHV-1 neg 4 withFD- BVDV pos gmBoHV-1 Unvaccinated BoHV-1/Mh² BoHV-1 neg 4 BVDV posVaccinated BVDV/Mh³ BoHV-1 pos 4 with FD- BVDV neg gmBoHV-1 UnvaccinatedBVDV/Mh³ BoHV-1 pos 4 BVDV neg ¹Indicate serological status at the timeof vaccination, BoHV-1 pos and BVDV pos indicate prior exposure toeither BoHV-1 or BVDV as determined by the presence of antibody to therespective viruses in the serum collected prior to vaccination.²Challenge with a BoHV-1 field strain Q3932 delivered via aerosol at 14days post-vaccination, followed by challenge with M. haemolytica 5 dayslater as described in Example 4. ³As for 2 except BVDV field strain wasused instead of BoHV-1.

Environmental swabs were taken from various locations in and around theanimal house to test for the presence of the GM virus persisting outsideits natural host (cattle) on each day that animal samples werecollected.

Prior to vaccination (Day 0) blood (20 ml), nasal swabs and nasal tamponswabs were collected from all cattle. The rectal temperatures and weight(combined pair weight) of all cattle was also recorded. Groups were thenvaccinated intra-nasally with the freeze-dried gmBoHV-1 that wasreconstituted immediately prior to instillation. It was planned toadminister the vaccine using a commercial applicator, however, due toproblems in getting this to work, the vaccinations were delivered usinga syringe as performed in previous research.

For 7 days following vaccination (Day 1-7) nasal swabs were collectedfrom all animals and clinical assessments recorded for all animals.

All cattle were challenged with the BoHV-1 strain Q3932 on Day 14post-vaccination as described below. Prior to the challenge, nasal swabscollected and clinical assessments recorded for all animals. The cattlewere then challenged with intra-nasal instillation of 10⁷ TCID₅₀ BoHV-1strain Q3932. After BoHV-1 challenge nasal swabs were collected from allcattle and clinical assessments made on a daily basis (Day 15-18) usingthe scoring method described in Table 3.

All cattle were challenged with M. haemolytica on Day 18post-vaccination. Prior to the challenge nasal swabs were collected fromall animals and rectal temperatures, respiration rates and weightsrecorded for all animals. The cattle were then challenged with anintra-nasal instillation of approximately 5×10⁹ cfu of M. haemolytica.After this secondary challenge nasal swabs were collected from allcattle and clinical assessments made on a daily basis (Day 19-26) usingthe scoring method described in Table 3.

The trial concluded on Day 35 post-vaccination. At this time blood (20ml), nasal swabs and nasal tampon swabs were collected from each animal.After the collection of these samples all animals were euthanized andtissue samples (heart, long, kidney, spleen, muscle, liver, brain andtrigeminal ganglia) were collected. Carcases were disposed of via deepburial.

Example 6 Reversion to Virulence

The aim of this trial was to determine if the serial passage of thegmBoHV-1 through multiple groups of cattle would show evidence ofincreasing virulence.

Environmental swabs were taken from various locations in and around theanimal house to test for the presence of the GM virus persisting outsideit natural host (cattle) on each day that animal samples were collected.

On the day prior to vaccination (Day 0) blood (20 ml), nasal swabs andnasal tampon swabs were collected from the cattle. The rectaltemperatures and weight (combined pair weight) of all cattle was alsorecorded.

The cattle (2) were then vaccinated intra-nasally with the freeze-driedgmBoHV-1 which was reconstituted immediately prior to instillation.Following vaccination (Day 1-7) nasal swabs were collected form theseanimals and clinical assessments recorded for the animals on a dailybasis.

Each passage experiment concluded on Day 14 post-vaccination. At thistime blood (20 ml) nasal swabs and nasal tampon swabs were collectedfrom each animal. After the collection of these samples all animals wereeuthanized and tissue samples (heart, lung, kidney, spleen, muscle,liver, brain and trigeminal ganglia) were collected. Carcases weredisposed of via deep burial.

To complete the in vivo passage of the gmBoHV-1 virus, virus wasreisolated from nasal swabs of the vaccinated cattle and used toreinfect two BoHV-1 naïve cattle. This process was repeated four timesas described above. Note that only the first passage was completed usingfreeze-directed gmBoHV-1.

Example 7 Excess Dose

The aim of this trial was to determine if the administration of anexcessive dose (ED) of gmBoHV-1 would be deleterious to the vaccinatedanimal.

Environmental swabs were taken from various locations in and around theanimal house to test for the presence of the GM virus persisting outsideits natural host (cattle) on each day that animal samples werecollected.

Prior to vaccination (Day 0) blood (20 ml), nasal swabs and nasal tamponswabs were collected from all cattle. The rectal temperatures and weight(combined pair weight) of all cattle was also recorded. Ed-Groups 1 to 3were then vaccinated intra-nasally with the freeze-dried gmBoHV-1 whichwas reconstituted immediately prior to instillation as described below.It was planned to administer the vaccine using commercial applicator,however, due to problems in getting this to work, the vaccinations weredelivered using a syringe as performed in previous research.

-   ED-Group 1: Vaccinate cattle (4) using intra-nasal instillation with    (10⁵⁻⁶ TCID₅₀) 1 ml of vaccine per nostril;-   ED-Group 2: Vaccinate cattle (4) using intra-nasal instillation with    (10⁶⁻⁷ TCID₅₀) 1 ml of vaccine per nostril;-   ED-Group 3: Vaccinate cattle (4) using intra-nasal instillation with    (10⁷⁻⁸ TCID₅₀) 1 ml of vaccine per nostril.

For seven days following vaccination (Day 1-7) nasal swabs werecollected from all animals and clinical assessments recorded for allanimas. The trial concluded on Day 14 post-vaccination. At this timeblood (20 ml), nasal swabs and nasal tampon swabs were collected fromeach animal. After the collection of these samples all animals wereeuthanized and tissue samples (heart, lung, kidney, spleen, muscle,liver, brain and trigeminal ganglia) were collected.

Example 8 Genetic Stability of the gmBoHV-1

Restriction Enzyme Profiles on Back Passaged gmBoHV-1 Prototype Vaccine

From the nasal swabs collected throughout the Pen Trials, treatmentgroup representatives were identified for viral isolation via mammaliancell culture (CRIB-1 cells). The presence of the bacterial artificialchromosome (BAC) within the backbone of the gmBoHV-1 enables theisolation and purification of plasmid DNA via bacterial replication toincrease DNA yield.

Cell Culture

Confluent monolayers (70%) of CRIB-1 cells were prepared in six wellplates for infection. The media was removed and the monolayers werewashed with sterile phosphate buffered saline (PBS). To the washedmonolayers 1 mL of the nasal swabs (in PBS and five times PSF) wereadded and incubated at 27° C. in 5% v/v CO₂ for 1 hour. The inoculumswere removed and the cells washed once with 1 mL of PBS then allowed torecover in 3 mL of fresh media with PSF for 5 h at 37° C. and 5% CO₂.

At 6 h post infection, the monolayers were washed with 1 mL PBS then 1mL of Total DNA Lysis buffer (with fresh Proteinase K) was added to themonolayers and incubated for 4 hours at 37° C. This lysed the cells insitu releasing total DNA from the CrIB-1 cells and the replicatingpBACBHV1E2s viral vaccine candidate. Harvesting the total DNA at thisearly stage of the infection ensures that some BAC DNA will be circularand suitable for transformation into bacteria for clonal replication.

Total DNA Extraction and Transformation

The total DNA was purified by using phenol/chloroform extraction andabsolute alcohol to precipitate. The dried DNA pellet was thenresuspended in 50 μl sterile high pure water (18 MΩ) at room temperaturefor 2 h. The volumes of each extraction varied depending upon theviscosity of the initial suspension of DNA. Transformation was achievedthrough electrophoration of 10 μl of the total DNA into 20 μl of DH10BElectroMax competent cells (Invitrogen) and selection on bacterialplates containing 12.5 μg/ml Chloramphenicol. three colonies from eachplate were picked into 50 mL LB broth containing 12.5 μg/mlChloramphenicol and grown over 18 h at 37° C. with gentle shaking. TheBAC DNA was extracted from these cultures using a modified alkalinelysis protocol (based upon the Roche high pure plasmid isolation kit formini preparations). The bacterial cells were pelleted to removed thebroth and resuspended in a Tris buffer. These cells were lysed torelease the plasmid DNA which was then purified, removing the remainingproteins and slats. The pelleted DNA was dried and resuspended in 60 μlof 10 mM Tris-HCl (pH 8.5) overnight at 4° C.

Restriction Enzyme Profiles

The multiple clones from each isolate were analyzed for gross mutationsvia RE profile comparisons with the vaccine candidate (not back passagedvia animals) and the parent viral vector (the pBACHBV1 37). 5 μl of thealkaline lysis prepared BAC DNA was digested for 4 h in a 20 μl totalvolume for enzymes Hindlll and Sall, (NEB) according to themanufacturer's instructions.

Field Inversion Gel Electrophoresis (FIGE) was used to separate andvisualize the band profiles for the digested BAC DNA. The 20 μl digestedBAC DNA was loaded onto 0.7% agarose gels in 0.5 times TBE buffer (bothwith ethidium bromide) and run under the conditions for program 3 on theFIGE apparatus (BioRad) targeting the molecular weight size range of5-100 Kb with a run time of 16 h.

Example 9 In Vitro Capacity of the gmBoHV-1 to Infect, Replicate andExpress the Transgene in Mammalian Cell-Lines

There was no distinguishable difference between the parent BoHV-1 andthe GM BoHV-1 in terms of the appearance of CPE. The CPE visualized wastypical of BoHV-1 for both viruses. Typically, the amount of virus(deduced from the CT values) in both the BoHV-1 or gmBoHV-1 infectedcell lines at 24 h post infection were similar (FIGS. 1A to C). For theHaCaT and CRIB-1 cell lines this was statistically significant, i.e.there was no significant difference in the amount of virus detectedbetween the parent and GM (P<0.05 two tailed unpaired t test). For theremaining cell lines the difference in CT values between the parent andGM infected cells was no more than 2.5 and neither virus wasconsistently detected at a higher level in every cell line.

It was established that the insertion of the transgene (synthetic BVDVE2) into the parent BoHV-1 did not alter the capacity of the virus toinfect and replicate in the various mammalian cell lines tested whencompared to the parent virus. Interestingly, all of the cell linestested were susceptible to BoHV-1 (both modified and unmodified)infection.

Example 10 Transmission of the gmBoHV-1 from Cattle to Other Ruminants

A pen trial was conducted to determine if either the host range ofBoHV-1 or the capacity of BoHV-1 to transmit to other ruminants had beenaltered as a result of the genetic modifications made. The pen trialincluded a series of sentinel cattle, sheep and goats placed at variousdistances from the vaccinated cattle.

At the commencement of the pen trial, it was noted that some of thecattle (particularly those in Sentinel Group A) had nasal discharge.There was also indications that some of the cattle had diarrhea. Whileit was not optimal to proceed with the trial it was not feasible topostpone the trial until these signs disappeared. These clinical signswere apparent in most of the cattle groups at some time throughout thetrial—some animals appeared to have signs throughout the trial.

Testing of the nasal swab extracts from Day 0 of all animals with theBRD multiplex assay (FLOT.219) did not identify any samples containing,Bovine herpesvirus 1, bovine respiratory syncytial virus, bovineparainfluenza virus 3 or bovine pestivirus. Standard PCR assays for fourgenera of paramyxoviruses, adenovirus and enterovirus were alsonegative. The absence of BoHV1 and pestivirus was most important forthis trial as the presence of either virus would have made it extremelydifficult to interpret the results of the trial and may have caused thetermination of the experiment.

Virus isolation attempts from the Day 0 nasal swab samples wasinteresting as there appeared to be some cytopathic effect (CPE) in thecells, indicating that a virus was present. However, attempts to passagethese supernatants were not successful. Further attempts to identify ifan infectious agent was associated with these signs were done bystaining cells infected with the nasal swab from the animal with themost persistent clinical signs (Animal 377 from Sentinel A) withfluorescently labeled antibodies to the following bovine viruses;adenovirus 3, coronavirus, pestivirus, parainfluenza virus, respiratorysyncytial virus, parvovirus and reovirus. The Day 0 nasal swab extractfor Animal 377 was then tested using a standard PCR and an ampliconconsistent with the expected size was obtained. However, sequencing ofthis amplicon indicated it was a non-specific amplification product.While the antibody staining appeared to be specific the identity of thepathogen remains unknown. Analysis of these samples with all moleculartests available in our lab did not identify the causative agentresponsible for the observed clinical signs. Based on previousapplication of these tests it is considered highly unlikely the agentresponsible was closely related to either BoHV-1 or BVDV and thus shouldnot interfere with the interpretation of serological data for theseviruses.

No gmBoHV-1 was detected either by RT-PCR or virus isolation from any ofthe sheep or goats from the three sentinel groups. Similarly no GM viruswas detected either by RT-PCR or virus idolization from any of thecattle held in the Sentinels groups A, B or C at anytime during thetrial.

GM virus was detected in all of the vaccinated cattle. Typically, if asample was positive for virus isolation (culture) it was positive byRT-PCR. The best recovery of virus was from an animal where nasal swabswere positive by RT-PCR from Day 1 to Day 6 and culture positive on Days3 and 4.

Contact transmission of the GM virus was detected in two of the fourpairs of animals.

Based on the RT-PCR amplifications of GM virus from nasal swabs, thepeak period for virus replication in the vaccinated cattle was Day 3. GMvirus was detected in three of the four vaccinates on Day 3.

To determine if the GM virus was able to persist outside the host(cattle) environmental swabs were collected from various surfaces at thetrial site throughout the experiment. The extracts from these swabs werethen tested for the presence of the GM virus using isolation in cellculture and RT-PCR. No GM virus was detected in any of the environmentalswabs taken throughout the experiment.

Testing of the sera collected on Day 0 and Day 28 from all of the trialanimals demonstrated only the vaccinated cattle developed detectableantibodies to BoHV-1 in the Day 28 samples.

On the basis of the results from this trial, there was no transmissionof the GM virus to other species (sheep and goats). Similarly,transmission of the GM virus over distances (>2 m) did not occur tocattle housed in proximity to the vaccinated cattle. Transmission of theGM virus to cattle housed with the vaccinated cattle did occur, thoughnot at a high frequency.

The results of the trial did not support any alteration of the hostrange for the GM virus. Although some transmission did occur to thenatural host (cattle), it was infrequent and maybe independent of thelevel of replication in the vaccinated animal. Added to this, no GMvirus was detected outside the natural host of the virus inenvironmental swabs collected throughout the experiment. Together, thesedata demonstrate that the risk of releasing the GM virus into theenvironment is minimal.

Example 11 Reactivation of gmBoHV-1

At the completion of each trial nasal swabs were collected fromvaccinated and unvaccinated cattle. Total nucleic acids were extractedfrom these swabs and the presence of the gmBoHV-1 tested for usingreal-time PCR assays targeting BoHV-1 and the E2 transgene. All of thesesamples were negative by both PCR assays. On this basis it is reasonableto conclude that no reactivation occurred prior to the completion of thetrial.

Example 12 Persistence and Stability of the GM BoHV-1

No differences could be determined between the stability of the GMBoHV-1 and the parent virus in field conditions. The license stipulatedthat the trial was conducted in a PC1 animal house where exposure todirect sunlight was not possible. Exposure to sunlight in true fieldconditions would likely increase the instability of both the GM andparent virus due to UV light.

Example 13 Residual Gene Products

No gmBoHV-1 was detected in any of the tissues tested for the animalsusing real time PCR. While it was considered unlikely that any geneproducts would be present a Western blot analysis was conducted on totalprotein extracts from the tissues of this animal. Based on these resultsit is unlikely that the GM virus or gene products it expresses persistin the tissues of vaccinated/infected animals.

It was not unexpected that the tissue samples of the animals werenegative for the presence of both the GM virus and transgene products.Of the tissues tested, the presence of gmBoHV-1 was only expected in thetrigeminal ganglia (TG) as this is the site where the parent virus isexpected to form a latent infection. The failure to detect virus in theTG cold indicate that the gmBoHV-1 is unable to establish latentinfection. Alternatively, the detection of the gmBoHV-1 in the TG maybedifficult as only a few cell bodies in the ganglion are likely to carrythe virus—thus the likelihood of successful detection depends on theamount of tissue processed and test sensitivity.

Example 14 Efficacy of a Freeze-Dried gmBoHV-1 Preparation

The efficacy of the gmBoHV-1 prototype vaccine as a freeze driedpreparation (FD-gmBoHV-1) was compared to gmBoHV-1 as a wet preparationin vaccination/challenge trials. A group of cattle vaccinated withRhinogard were included in this trial for comparison. Table 3illustrates the virus detection results for all vaccinated andunvaccinated cattle from Day 0 (day of vaccination) to Day 7. Generally,the FD-gmBoHV-1 and gmBoHV-1 vaccines were shed at the most consistentrates on Day 3 with high levels of virus detected by PCR and virusconsistently isolated (Table 6). The majority of cattle vaccinated withgmBoHV-1 were positive by both PCR assays on three or more days. Theexception to this was an animal (designed 581) that was positive only onDay 3 post-vaccination.

No adverse clinical signs were observed in either the vaccinated orunvaccinated groups. BoHV-1 was detected in three of the unvaccinatedanimals on three occasions during this phase of the experiment. Thevirus detected was not the gmBoHV-1 as the PCR assay specific for the E2transgene was negative for all animals. In addition the PCR results forBoHV-1 were weakly positive indicating that the results were due tocontamination of the sample. This is most likely to have occurred duringpost-handling of the samples at the laboratory for those positivesamples on Day 0.

There are some samples positive for the gmBoHV-1 on Day 0. These arelikely due to contamination within the vaccination group as it was notlogistically possible to decontaminate all surfaces between animalsreceiving the same treatment. Similarly animals were held within thecrush in pairs as this typically made them more settled, thus transferfrom the initial member of the pair to the other before the secondanimal was swabbed cannot be excluded.

To minimize the likelihood of any cross contamination between groups,groups were always processed in the following order; unvaccinatedcontrols, Rhinogard vaccinated, followed by gmBoHV-1. In addition, theanimal handling area including the crush, was decontaminated after theRhinogard vaccinated group.

The BoHV-1 positive result for the unvaccinated animal 549 on Day 5could have been due to transmission from the Rhinogard vaccinated group.Animal 549 was housed in pens adjacent to the Rhinogard vaccinate groupsand as a result had to pass these animals on the way to the crush area.While animals were closely monitored during this process to preventdirect contact it is still feasible that animal 549 may have inhaledvirus containing material while passing the Rhinogard pens as theanimals tend to investigate the environment during this movementprocess. That the virus did not infect 549 and was only detected on Day1 support that it was an environmental contamination rather thantransmission of the virus from an infected/vaccinated animal. The dataare shown in Table 6.

TABLE 6 Animal PCR Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 IDAssay PCR PCR PCR VI PCR VI PCR PCR PCR PCR Vaccinated with freeze-driedgmBoHV-1 543 BHV − + +++ Neg +++ Pos ++ +++ + ++ 38.6-40.1 E2s − + ++++++ ++ +++ + ++ 561 BHV − + +++ Neg ++ Pos ++ +++ − +++ 38.3-39.7 E2s− + +++ ++ ++ +++ − +++ 577 BHV − − ++ Neg ++ Pos − − + +++ 38.4-39.4E2s − − ++ ++ − − + +++ 581 BHV − − − Neg ++ Pos − − − − 38.8-39.7 E2s −− − ++ − − − − 540 BHV + + +++ Neg +++ Pos +++ +++ +++ +++ 38.5-39.2E2s + + +++ +++ +++ +++ +++ +++ 553 BHV − ++ +++ Neg − Pls ++ − +++ ++38.5-39.6 E2s − ++ +++ − ++ − +++ ++ 564 BHV − ++ − Neg +++ Pos ++ +++++ − 39.2-39.8 E2s − ++ − +++ ++ +++ ++ − 565 BHV + + + Neg ++ Pos + ++− − 39.1-40.6 E2s + + + ++ + ++ − − Vaccinated with gmBoHV-1 574 BHV + ++++ Pos +++ Pos ++ ++ ++ + 38.5-39.7 E2s + − +++ +++ ++ ++ ++ + 584BHV + + +++ NA +++ NA +++ +++ ++ − 38.9-39.8 E2s + + +++ +++ +++ +++ ++− 595 BHV − − +++ NA ++ NA ++ ++ − − 38.7-39.5 E2s − − +++ ++ ++ ++ − −596 BHV − − +++ NA +++ NA ++ + + − 38.9-39.7 E2s − − +++ +++ ++ + + −Vaccinated with Rhinogard 550 BHV − − − NA − NA ++ +++ ++ +++ 38.7-39.5E2s − − − − − − − − 551 BHV − + +++ NA +++ NA +++ +++ +++ +++ 38.7-39.1E2s − − − − − − − − 555 BHV + − +++ NA ++ NA ++ +++ +++ +++ 38.6-39.6E2s − − − − − − − − 570 BHV − − ++ NA ++ NA ++ ++ ++ +++ 38.5-39.4 E2s −− − − − − − − Unvaccinated 549 BHV − − − NA − NA − + − − 38.8-39.8 E2s −− − − − − − − 572 BHV − − − NA − NA − − − − 38.2-39.6 E2s − − − − − − −− 590 BHV + − − NA − NA − − − − 38.7-39.6 E2s − − − − − − − − 591 BHV +− − NA − NA − − − − 38.5-39.2 E2s − − − − − − − − 546 BHV − − − NA − NA− − − − 38.5-39.2 E2s − − − − − − − − 583 BHV − − − NA − NA − − − −38.5-39.8 E2s − − − − − − − − 586 BHV − − − NA − NA − − − − 38.5-39.5E2s − − − − − − − − 587 BHV − − − NA − NA − − − − 38.6-39.2 E2s − − − −− − − −

-   -   Vaccination phase: virus detection and virus isolation results        for vaccinated and unvaccinated cattle. Cattle were vaccinated        with gmBoHV-1 (Wet GM), freeze-dried gmBoHV-1 (FD-GM), Rhinogard        (RG) or not vaccinated as part of pen trial to assess the        efficacy of the FD-GM compared to Wet GM. following extraction        of DNA from nasal swabs, the samples were tested using real-time        PCR assays (P) specific for the gmBoHV-1 vector (BHV) and BVDV        E2 transgene (E2). PCR results are expressed as, very strong        (++++, Ct value <20), strong (+++, Ct value >20 but <30), weak        (++, Ct value >30 but, 35), very weak (+, Ct value >35 but <40)        or negative (Pos), virus not recovered (Neg) or not attempted        (NA). The temperature (° C.) range for each animal from Day 0 to        7 are shown below the animal number. All temperatures recorded        were below 39.5° C. in the 7 days following vaccination, with        the exception of, 581 Day 1 39.7° C.; 596 Day 7 39.7° C.; 565        Days 3 and 5 39.7° C. and 39.6° C., respectively.

Fourteen days after the initial vaccination of the treatment groups thecattle were challenged with either BoHV-1 strain Q3932 or the BVDVstrain MD74. BoHV-1 was detected in the nasal swabs of two animalscollected prior to administration of the challenge viruses (Table 7).The nasal swabs were negative for the E2 transgene which indicated thatthe detected virus was not the gmBoHV-1. There is no apparent source ofthis virus as all animals vaccinated with Rhinogard were virus negativeindicating that the likely source was in post-collection handling of thesamples. High levels of BoHV-1 challenge strain Q3932 were detected inall BoHV-1 challenged animals (Table 7) at three to four dayspost-challenge (Days 17 and 18).

Biosecurity measures were implemented to prevent either the transmissionof BoHV-1 to the BVDV challenge groups or the transmission of BVDV tothe BoHV-1 challenge groups. Despite this there was significantinfection of the BVDV challenge group with BoHV-1 (Table 7). The sourceof this cross-infection is not readily apparent. While no studies havebeen conducted to specifically assess the capacity of the AustralianBoHV-1 strains to spread between cattle, it is generally accepted thatclose contact is required for transmission to occur. It may be that themajority of trials conducted that underpin this conclusion werevaccination trials and as a result there has been no observedtransmission of the vaccine strain (BoHV-1 strain V155). However, in thechallenge phase of these experiments all animals are challenged thusthere was no opportunity to assess the transmission of the challengestrain to naïve animals. On this basis it would seem that the challengestrain Q3932 is more readily transmitted to other cattle by means otherthan close contact.

TABLE 7 Day 14 15 16 17 18 19 20 21 22 23 24 25 Animal ID Assay P P P PP P P P P P P P Vaccinated with freeze dried gmBoH-1 Challenge 543 BHV −++ +++ ++++ +++ +++ +++ +++ ++ − − − 37.9-39.2 E2s − − − − − − − − − − −− Mh − +++ +++ +++ +++ + ++ + 561 BHV − +++ ++++ +++ ++++ ++++ +++ ++++++ +++ ++ ++ 38.1-39.1 E2s − − − − − − − − − − − − Mh − ++ +++ +++ ++++ − ++ 577 BHV − ++ +++ +++ ++++ ++++ ++++ +++ +++ +++ +++ ++ 39.6-38.9E2s − − − − − − − − − − − − Mh − ++ + ++ − − − − 581 BHV − +++ +++ ++++++++ ++++ ++++ +++ +++ + ++ − 38.3-40.2 E2s − − − − − − − − − − − − Mh −++ ++ +++ ++ + + ++ 574 BHV − ++ ++++ +++ +++ +++ ++++ +++ +++ +++ ++ +38.0-39.4 E2s − − − − − − − − − − − − Mh ++ +++ +++ ++ ++ +++ ++ 584 BHV− +++ ++++ ++++ ++++ +++ +++ +++ ++ − − + 38.0-39.1 E2s − − − − − − − −− − − − Mh − +++ +++ +++ ++ ++ − + 595 BHV − ++++ +++ ++++ +++ ++++ ++++++ +++ ++ + − 38.0-39.5 E2s − − − − − − − − − − − − Mh − ++ +++ ++++++ + + ++ 596 BHV − +++ +++ ++++ +++ ++++ +++ +++ ++ ++ − + 38.2-39.9E2s − − − − − − − − − − − − Mh − +++ ++ ++ ++ + − − Unvaccinated andBoHV-1 Challenge 549 BHV + ++ +++ +++ ++++ ++++ ++++ ++++ +++ +++ ++++++ 38.4-39.5 E2s − − − − − − − − − − − − Mh − ++ +++ +++ +++ +++ ++++++ 572 BHV − +++ +++ ++++ ++++ ++++ ++++ ++++ +++ +++ ++ ++ 38.3-40.2E2s − − − − − − − − − − − − Mh − +++ +++ +++ +++ +++ ++ ++ 590 BHV − ++++++ +++ ++++ ++++ +++ ++++ ++++ +++ ++ ++ 38.2-40.1 E2s − − − − − − − −− − − − Mh − ++ ++ ++ +++ +++ ++ ++ 591 BHV − +++ ++++ +++ ++++ ++++++++ ++++ +++ +++ +++ ++ 38.5-39.9 E2s − − − − − − − − − − − − Mh − ++++++ +++ +++ +++ +++ ++ Rhinogard vaccinated/BoHV-1 Challenge 574 BHV −++ ++++ +++ +++ +++ ++++ +++ +++ +++ ++ +   38-39.4 E2s − − − − − − − −− − − − Mh − ++ +++ +++ ++ ++ +++ ++ 584 BHV − +++ ++++ ++++ ++++ ++++++ +++ ++ − − +   38-39.1 E2s − − − − − − − − − − − − Mh − +++ +++ +++++ ++ − + 595 BHV − ++++ +++ ++++ +++ ++++ +++ +++ +++ ++ + −   38-39.5E2s − − − − − − − − − − − − Mh − ++ +++ +++ +++ + + ++ 596 BHV − +++ +++++++ +++ ++++ +++ +++ ++ ++ − + 38.2-39.9 E2s − − − − − − − − − − − − Mh− +++ ++ ++ ++ + − Mh − ++ + + + + − + 570 BHV − ++ ++ +++ +++ +++ ++++++ +++ +++ +++ ++ 38.7-39.4 E2s − − − − − − − − − − − − Mh − ++ ++ ++++ ++ − − Vaccinate with freeze-dried gmBoHV-1/BVDV Challenge 540 BHV +− − − − +++ ++++ +++ ++++ +++ − +++ 38.4-39.5 E2s − − − − − − − − − − −− BVDV + − − − − − − − − − Mh − ++ + − − − + − 553 BHV − − ++ − ++ +++++ +++ +++ +++ +++ +++ 38.4-39.3 E2s − − − − − − − − − − − − BVDV − − −− − − − − − − − Mh − − − − − − − ++ − 564 BHV − − ++ − ++ +++ ++++ ++++++++ ++++ ++++ ++++ 38.9-39.5 E2s − − − − − − − − − − − − BVDV − − − − −− − − − − − Mh − + + + ++++ − + − 565 BHV − − − ++++ − ++ +++ ++++ ++++++++ +++ +++   39-39.7 E2s − − − − − − − − − − − − BVDV − − − − − − − −− − − Mh − − − − − − ++ Unvaccinated/BVDV Challenge 546 BHV − − − + + +++++ +++ +++ ++++ +++ +++ 38-39 E2s − − − − − − − − − − − − BVDV − − − +− − − − − − − Mh − − − + − − ++ ++ 583 BHV − − − − ++ +++ +++ +++ +++++++ +++ ++++ 38.1-39.2 E2s − − − − − − − − − − − − BVDV − − − − − − − −− − − Mh − ++ + − + ++ +++ +++ 586 BHV − + − − ++ +++ +++ +++ ++++ +++++++ ++++ 38.5-39.5 E2s − − − − − − − − − − − − BVDV − − − − − − − − − −− Mh − + − ++ + − +++ +++ 587 BHV − − − − + ++ +++ +++ ++++ ++++ +++++++ 38.0-38.9 E2s − − − − − − − − − − − − BVDV − − − − − − − − − − − Mh− + − − − + − −

-   -   Challenge phase virus detection, virus isolation and Manhiemia        haemolytica detection results for vaccinated and placebo cattle        after challenge. Day 14 post vaccination cattle were challenged        with either BoHV-1 (10⁷ TCID₅₀) or BVDV, a subsequent challenge        of M. haemolytica (6.8×10⁹CFU) was administered to all cattle on        Day 18. Following extraction of DNA from nasal swabs, the        samples were tested using real-time PCR assays (P) specific for        the BoHV-1 challenge strain Q3932 (HBV), BVDV E2 transgene (E2),        BVDV challenge strain (BVDV) or M. haemolytica (Mh). PCR results        are expressed as, very strong (++++, Ct value <20), strong (+++,        Ct value >20 but <30), weak (++, CT value >30 but <35), very        weak (+, Ct value >35 but <40) or negative (−). The temperature        (° C.) ranges for each animal from Day 14-25 are shown below the        animal number. One Animal 546 had a Ct value of 36.7 for BHV        only at the end of the trial. The BHV PCR detects both the        gmBoHV-1 and the challenge strain of BoHV-1.

Following the administration of the two challenge pathogens, animalswere assessed for clinical signs on a daily basis. No clinical signswere recorded prior to the viral challenge from Day 14 to Day 18. Afterthe second phase of the challenge with the M. haemolytica clinical signswere apparent in many of the groups. Generally, the clinical signsobserved were mild. No elevated temperature, lose of appetite,alteration of respiratory rate or coughing were recorded at any timeduring the challenge phase.

Sera samples were collected from all animals at Day 0 (vaccination). Day14 (viral challenge) and at the end of the trial immediately prior toeuthanasia. The sera were tested for the presence of antibodies toBoHV-1 and BVDV using commercially available ELISA tests. The results ofthese tests are shown in Table 8. On Day 0, six of the cattle werepositive for antibodies to BoHV-1. The cattle (60) were sourced from asingle property and were all around the same age. Sera collected fromall cattle was tested and were negative for antibodies specific to BVDV.However, the sera from 23 of the 60 cattle were positive for antibody toBoHV-1 from the same herd previously with levels considered to be morenormal with less than 10% positive for BoHV-1. Due to the highseroprevalence and the relatively young age of the cattle (weanedapproximately 8 weeks prior to arrival), it was considered likely thatthe high prevalence of BoHV-1 positives was due to maternal antibody. Ifmaternal antibody was responsible then it would be expected that theamount of antibody present in the serum would decline overtime. As aresult the cattle were retested for the presence of BoHV-1 antibodies ona weekly basis. Between the period of the first test and the third testthree of the cattle went from positive to negative, one from positive todoubtful, 15 indicated reducing levels of antibody and four remainedpositive with steady levels of antibody. One animal appeared to developantibodies to BoHV-1 (Number 598), however, it was seronegative whentested later.

At the commencement of the trial, all cattle positive for BoHV-1antibodies (Table 8) had reduced levels compared to the previous testwhich again supports the presence of material antibodies in theseanimals.

As would be expected, all of the cattle vaccinated with the gmBOHV-1sero-converted with respect to BoHV-1 by the end of the trial.

All cattle remained sero-negative to BVDV throughout the trial (Table8). This was not expected as those animals challenged with BVDV wereexpected to sero-covert to BVDV. However, in the context of the virusdetection results it is not surprising that no sero-conversion wasdetected as the BVDV strain used does not appear to have replicated inthe unvaccinated animals. Animal 546 was the only animal PCR positivefor BVDV (on Day 18) four days post challenge with BVDV while this couldbe considered a long time for virus to persist in the nasal cavitywithout infecting and replicating, if replication did take place, thenit must have been at a very low level as the animal did no sero-convertnor was virus detected on any other day. The serology results supportthe virus detection results for BVDV indicating that the BVDV strain didnot infect nor replicate in these animals.

TABLE 8 END OF Animal DAY 0 DAY 14 TRIAL ID BHV BVDV BHV BVDV BHV BVDVF/D GM 581 Pos Neg Neg Neg Pos Neg Vaccination 577 Pos Neg Neg Neg PosNeg 543 Neg Neg Neg Neg Pos Neg 561 Neg Neg Neg Neg Pos Neg GM 595 NegNeg Pos Neg Pos Neg Vaccination 596 Neg Neg Pos Neg Pos Neg 574 Pos NegPos Neg Pos Neg 584 Pos Neg doubt Neg Pos Neg Un- 590 Neg Neg Neg NegPos Neg vaccinated 591 Neg Neg Neg Neg Pos Neg 549 Neg Neg Neg Neg PosNeg 572 Neg Neg Neg Neg Pos Neg Rhinogard 570 Neg Neg Neg Neg Pos Negvaccination 555 Neg Neg Pos Neg Pos Neg 551 Neg Neg Pos Neg Pos Neg 550Neg Neg Neg Neg Pos Neg F/D GM 553 Pos Neg Pos Neg Pos Neg Vaccination540 Pos Neg Pos Neg Pos Neg 564 Neg Neg Neg Neg Pos Neg 565 Neg Neg PosNeg Pos Neg Un- 586 Neg Neg Pos Neg Pos Neg vaccinated 587 Neg Neg NegNeg Pos Neg 583 Neg Neg Neg Neg Pos Neg 546 Neg Neg Neg Neg Pos Neg

-   -   Serological status of trial cattle to Bovine herpesvirus 1 (BHV)        or bovine viral diarrhea virus (BVDV) at various stages        throughout the vaccination trial. Sera samples from all cattle        were tested using the Pourquier (Registered Trade Mark) ELISA        IBR-IPV Serum and Milk for detection of serum antibodies to HBV        and Pouriquier (Registered Trade Mark) ELISA BVD-MD-BD P80        Antibodies for detection of serum antibodies to BVDV. The BHV        specific test will confirm prior infection with either wild-type        BoHV-1 or gmBoHV-1. The BVDV specific test will confirm prior        infection with wild-type BVDV, it does not detected antibodies        specific for the BVDV E2.

Example 15 Effects of Pre-Existing Immunity on Vaccine Efficacy

A possible risk of combining vaccines using genetic engineering is thatpre-existing immunity to either the vector or the transgene could reduceany effectiveness of the vaccination. For example, in the current study,if cattle have pre-existing immunity to BoHV-1, which is the vaccinevector, then this may prevent replication of the gmBoHV-1 vaccine andeither prevent or reduce the stimulation of any immunological responseto the BVDV E2 protein encoded by the transgene. If this was to occurthen the vaccinated cattle would not have the opportunity to benefitfrom the BVDV component of the vaccine, i.e. the cattle would still besusceptible to BVDV infection and development of disease.

To determine if pre-existing immunity could interfere with the efficacyof the prototype vaccine, trials were conducted using animals that wereantibody positive for either BoHV-1 or BVDV. The responses tovaccination with the gmBoHV-1 were then assessed in the two stagechallenge model used previously.

Effects of Pre-Existing Immunity to BoHV-1 on Vaccine Efficacy

Cattle determined to be positive for antibody specific to BoHV-1 werevaccinated with the gmBoHV-1. DNA isolated from nasal swabs for theseanimals were then tested using real-time PCR assays specific for BoHV-1and the E2 transgene. As expected all animals were negative for bothassays on Day 0. Of the animals vaccinated with the gmBoHV-1, virus wasconsistently detected via both PCR assays from Day 1 to Day 6post-vaccination for all animals except Animal 552.

No adverse clinical signs were observed in the vaccinated animals duringthe seven days post vaccination.

One of the unvaccinated animals (Animal 557) was positive for both PCRassays on Day 7. Attempts to isolate virus from this nasal swab wereunsuccessful indicating that the PCR result was due to crosscontamination of the sample either during sub-aliquoting or DNAextraction processes.

One of the vaccinated animals (Animal 556) was PCR positive for BoHV-1on Day 7 only. As the PCR assay for the E2 transgene is more sensitivethan the BoHV-1 assay, it would appear that this is a non-recombinant orwild-type strain of BoHV-1. No attempts were made to isolate virus fromthis sample. As use of BoHV-1 nucleic acids is wide spread within thelaboratory cross contamination of the sample during eithersub-aliquoting or DNA extraction appears the most likely cause of thisresult.

Fourteen days after the vaccination both the vaccinated and unvaccinatedgroups were challenged with BVDV. All cattle were negative by PCR forBoHV-1, E2 transgene and BVDV on Day 14. The cattle from both groupswere challenged with BVDV and swabbed for a further five days. On Day 18post-vaccination all cattle were challenge with M. haemolytica. UsingBVDV strain three of the eight cattle were positive on at least one dayof the challenge phase. This first animal was positive on Day 15 and thelast on Day 23, a further three of the unvaccinated animalssero-converted to BVDV. These results are indicative that the BVDVchallenge was successful.

Following the BVDV challenge all cattle were assessed for clinical signson a daily basis. No clinical signs were observed between Day 14 and Day18. Clinical signs observed from Day 19 to Day 25.

Serological results for these groups are shown in Table 9. As can beseen all animals except for 539 were positive for BoHV-1 on Day 0, it issuspected that this animal contained maternal antibodies that haddissipated prior to the commencement of this trial. This animal remainednegative for BoHV-1 antibodies over the course of the study. As shown inTable 8, three of the eight animals (all unvaccinated) sero-converted toBVDV. An additional three of the eight animals (one unvaccinated and twovaccinated) demonstrated clearly increasing antibody levels to BVDV bythe end of the trial. These results support the assertion that the BVDVchallenge was successful.

TABLE 9 END Animal DAY 0 DAY 14 OF TRIAL ID BHV BVDV BHV BVDV BHV BVDVVaccinated 552 Pos Neg Pos Neg Pos Neg 556 Pos Neg Pos Neg Pos Neg* 563Pos Neg Pos Neg Pos Neg* 576 Pos Neg Pos Neg Pos Neg Un- 539 Neg Neg NegNeg Neg Pos vaccinated 557 Pos Neg Pos Neg Pos Pos 562 Pos Neg Pos NegPos Neg* 589 Pos Neg Pos Neg Pos Pos

-   -   Serological status of trial cattle to bovine herpesvirus 1 (BHV)        or bovine viral diarrhea virus (BVDV) at various stages        throughout the vaccination trial. Sera samples from all cattle        were tested using the Pourquier (Registered Trade Mark) ELISA        IBR-IPV Serum and Milk for detection of serum antibodies to BHV        and Pourquier (Registered Trade Mark) ELISA BVD-MD-BD P80        Antibodies for detection of serum antibodies to BVDV. The BHV        specific test will confirm prior infection with either wild-type        BoHV-1 or gmBoHV-1. The BVDV specific test will confirm prior        infection with wild-type BVDV, it does not detect antibodies        specific for the BVDV E2. *Clear trend of increasing antibody        levels.

Together these results indicate that the vaccination of the cattle withthe gmBoHV-1 afforded protection to these cattle.

Effects of Pre-Existing Immunity to BVDV on Vaccine Efficacy (BoHV-1Challenge)

Cattle determined to be positive for antibody specific to BVDV werevaccinated with the gmBoHV-1. DNA isolated from nasal swabs for theseanimals were then tested using real-time PCR assays specific for BoHV-1and the E2 transgene. The results of these PCR analyses are shown inTable 10. As expected, all animals were negative for both assays on Day0. Of the animals vaccinated with the gmBoHV-1, virus was consistentlydetected via both PCR assays from Day 2 to Day 6 post-vaccination forall animals. Virus was isolated from all animals on Day 3. Together thisdata support good uptake of the vaccine. Vaccine virus was not detectedin any of the unvaccinated animals (Table 10).

No adverse clinical signs were observed in the vaccinated animals duringthe seven days post vaccination.

TABLE 10 Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Animal ID P P PP VI P P P P VI Vaccinated 559 BHV − − ++ ++ Pos ++ + ++ ++ 37.9-39.4E2s − − ++ ++ ++ + ++ ++ 593 BHV − − ++ ++ Pos ++ ++ ++ ++ 38.2-39.1 E2s− − ++ ++ ++ ++ ++ ++ 573 BHV − + ++ ++ Pos ++ ++ ++ ++ 38.6-39.7 E2s− + ++ ++ ++ ++ ++ ++ 580 BHV − + + ++ Pls ++ + ++ + 38.6-40.4 E2s − + +++ ++ + ++ + Unvaccinated 554 BHV − − − − NA − − − − 37.9-39.2 E2s − − −− − − − − 560 BHV − − − − NA − − − − 38.1-39.3 E2s − − − − − − − − 544BHV − − − − NA − − − − 38.4-40.3 E2s − − − − − − − − 594 BHV − − − − NA− +* − 33.28 − 37.8-40.3 E2s − − − − − +* − 32.55 −

-   -   Virus detection and isolation results for cattle with existing        immunity to BVDV. Vaccination phase: virus detection and virus        isolation results for vaccinated and unvaccinated cattle with        pre-existing immunity to BVDV. Cattle were either vaccinated        with freeze-dried gmBoHV-1 (FD-GM) or remained unvaccinated.        Following extraction of DNA from nasal swabs, the samples were        tested using real-time PCR assays (P) specific for the gmBoHV-1        vector (BHV) and BVDV E2 transgene (E2). PCR results are        expressed as, very strong (++++, Ct value <20), strong (+++, Ct        value >20 but <30), weak (++, CT value >30 but <35), very weak        (+, Ct value >35 but <40) or negative (−). Virus isolation was        also attempted for selected samples (VI), isolation results are        shown as virus recovered (Pos), virus not recovered (Neg) or not        attempted (NA). the temperature ranges (° C.) for each animal        from Day 0 to 7 are shown below the animal number. *Virus        isolation was attempted with no virus isolated, the ratios of        the BoHV-1 and E2s PCR results was also inconsistent with        previous results.

On Day 14 post-vaccination all cattle were challenged with BoHV-1 strainQ3932 as previously described. Nasal swabs were collected on a dailybasis and clinical signs recorded. On Day 18 all animals were challengedwith M. haemolytica. Nasal swabs were collected on a daily basis andclinical signs recorded to Day 25 post-challenge. DNA was extracted fromall nasal swabs.

Fourteen days after the vaccination both the vaccinated and unvaccinatedgroups were challenged with BoHV-1. All cattle were negative by PCR forBoHV-1 and the E2 transgene on Day 14. The cattle from both groups werechallenged with BoHV-1 strain Q3932 and swabbed for a further five days.On Day 18 post-vaccination all cattle were challenged with M.haemolytica. As expected, all animals were negative by PCR for the E2transgene. The Q2932 was consistently detected in all animals, however,there was a trend towards vaccinated animals shedding less virus over ashorter time period compared to unvaccinated. A similar trend is evidentfor the M. haemolytica PCR assay.

Following the BoHV-1 challenge, all cattle were assessed for clinicalsigns on a daily basis. No clinical signs were observed between Day 14and Day 18.

Serological testing of the cattle demonstrated sero-conversion withrespect to BoHV-1 at the end of the trial for all cattle challenged withBoHV-1 as would be expected (Table 11).

TABLE 11 DAY 0 DAY 14 TRIAL END Animal BHV BVDV BHV BVDV BHV BVDVVaccinated 559 Neg Pos Neg Pos Pos Pos 593 Neg Pos Pos Pos Pos Pos 573Neg Pos Neg Pos Pos Pos 580 Neg Pos Neg Pos Pos Pos Un- 554 Neg Pos NegPos Pos Pos vaccinated

-   -   Serological status of trial cattle to Bovine herpesvirus 1 (BHV)        or bovine viral diarrhoea virus (BVDV) at various stages        throughout the vaccination trial. Sera samples from all cattle        were tested using the Pourquier (Registered Trade Mark) ELISA        IBR-IPV Serum and Milk for detection of serum antibodies to BHV        and Pourquier (Registered Trade Mark) ELISA BVD-MD-BD P80        Antibodies for detection of serum antibodies to BVDV. The BHV        specific test will confirm prior infection with either wild-type        BoHV-1 or gmBoHV-1. The BVDV specific test will confirm prior        infection with wild-type BVDV, it does not detect antibodies        specific for the BVDV E2.        Pre-Existing Antibody to Either BVDV or BoHV-1 does not Prevent        Replication or Recovery of the gmBoHV-1 Vaccine Virus from        Vaccinated Animals

Overall the results support the delivery of multiple antigens from otherpathogens using a live viral vector. Further the results indicated thatimmune status of the host with respect to the vaccine vector will notnegatively effect vaccine performance.

Example 16 Reversion to Virulence

The use of live viral vaccines carries an inherent risk of the parentvirus increasing in virulence if it is transmitted from one animal toanother susceptible animal. In order to investigate if this was likelywith the gmBoHV-1 and also to assess the stability of the geneticmodifications made, the prototype vaccine was passaged four timesthrough immunologically naïve (with respect to BoHV-1 and BVDV) cattle.These passage experiments were conducted in parallel with othervaccination trials.

As the gmBoHV-1 was most consistently detected and isolated on Day 3post-vaccination, virus isolated at this time was used for thesubsequent passage. The first passage was from animal number 598.

DNA isolated from nasal swabs for these animals were then tested usingreal-time PCR assays specific for BoHV-1 and the E2 transgene. Allanimals were negative for both assays on Day 0. Of the animalsvaccinated with the gmBoHV-1 virus was consistently detected via bothPCR assays from Day 2 to Day 7 post-vaccination for all animals. Viruswas isolated from all animals on Day 3.

No adverse clinical signs were observed in the vaccinated animals duringthe seven days post vaccination. Mild elevated temperatures (>40° C.)were detected for some animals during the passages, however, these weresporadic and did not appear to be related to the presence of virus.

As expected the majority of the animals sero-converted to BoHV-1 at theend of the trial.

In summary, no evidence was found to support the increased virulence ofthe gmBoHV-1 during the passage experiments. Further the transgeneappeared to be very stable with no evidence found to indicate lossthrough any of the passages.

Together the results of the passage experiments indicated that the E2transgene is stable within the BoHV-1 genome. Similarly, no evidence wasfound of the gmBoHV-1 reverting to a more virulence phenotype. Thegenetic stability of the gmBoHV-1 was also investigating by examiningthe restriction endonuclease digestion patterns of genome DNA fromreisolated viruses.

Example 17 Excess Dose

To be economically viable, vaccines are typically supplied in multipledose formulations. A possible drawn back of these formulations is thepotential for adverse effects on the vaccinated animals if the vaccineis used as a higher than recommended does. To investigate the likelihoodof adverse effects if the gmBoHV-1 was administered at a higher thanrecommended dose a trial was conducted where cattle were vaccinated withvarious concentrations of the vaccine.

Three groups of cattle (four per group) were vaccinated in each nostrilwith, 10^(6.5) TCID₅₀ of the gmBoHV-1 (10× Dose), 10^(5.5) TCID₅₀ of thegmBoHV-1 (expected effective dose) or 10^(4.5) TCID₅₀ of the gmBoHV-1(0.1× expected effective dose). Cattle were monitored for clinical signsand nasal swabs taken on a daily basis following vaccination. DNAisolated from nasal swabs for these animals were then tested usingreal-time PCR assays specific for BoHV-1 and the E2 transgene.

Of the animals vaccinated with the gmBoHV-1, virus was consistentlydetected via both PCR assays from D2 to Day 7 post-vaccination for allanimals. Virus was consistently isolated from animals on Day 3 wereattempted.

No adverse clinical signs were observed in the vaccinated animals duringthe seven days post vaccination. Mild elevated temperatures (>40° C.)were detected fro some animals, however, these were sporadic and did notappear to be related to the presence of virus.

As expected, the majority of the animals sero-converted to BoHV-1 at theend of the trial. There was a trend towards more animals sero-convertingin treatments receiving high quantities of virus as might be expected.

There was no evidence for any deleterious effects on animals vaccinatedwith high doses of the gmBoHV-1. At lower doses of the vaccine thereappears to be less efficient up take of the vaccine based on thecapacity to detect virus in nasal swabs by PCR detection and/or virusisolation.

Example 18 Genetic Stability of gmBoHV-1

In this Example, the genetic stability of the gmBoHV-1 was evaluated byexamining the genetic profiles of vaccine strains isolated from animalsduring the serial passage of the prototype vaccine through cattle.

This assessment was made by first reisolating the gmBoHV-1 from nasalswabs collected from infected cattle. To proved evidence that repeatedpassage in cattle would not adversely affect the genetic stability ofthe prototype vaccine these analysis were conducted on virus recoveredform serial passage. The isolated and cloned genomes of randomlyselected clones were then examined by restriction endonuclease digestionwhich is a well accepted method for assessing the genetic stability ofherpesviruses. Two restriction endonucleases were used in the first wasHindlll which cuts the BoH V-1 genome an estimated 12 times and thusprovides a measure of any large scale genomic re-arrangements orrecombination events. The second enzyme used as Sall which cuts theBoHV-1 genome an estimated 45 times and thus provides a measure of anyfiner scale genomic re-arrangements or recombination events.

Viruses were recovered from nasal swabs collected on Day 3 and Day 7post-vaccination and restriction profiles determined Three were noobvious large or smaller scale re-arrangements based on the Hindll andSall profiles, respectively.

On the basis of the restriction endonuclease profiles of virusesisolated after the passages in cattle, there was no evidence of anygenetic variability. These data support the conclusion that the gmBoHV-1used to vaccinate cattle in this study is highly stable.

Those skilled in the art will appreciate that aspects enabled herein aresusceptible to variations and modifications other than thosespecifically described. It is to be understood that these aspectsinclude all such variations and modifications. Enabled herein are all ofthe steps, features, compositions and compounds referred to or indicatedin this specification, individually or collectively, and any and allcombinations of any two or more of the steps or features.

BIBLIOGRAPHY

-   Mahoney et al. (2002) Journal of Virology 76(13):6660-6668-   Narayanan et al. (1999) Gene therapy 6:442-447-   Orford et al. Nucleic Acids Research 28(18):e84-   Schumacher et al. (2000) Journal of Virology 74:11088-11098-   Snowden (1964) Australian Veterinary Journal 40:277-288

What is claimed is:
 1. A vaccine against at least one antigen from abovine pathogen, said vaccine comprising a bovine herpes virus-1(BoHV-1) genome from a low virulence BoHV-1, said BoHV-1 having geneticmaterial encoding the at least one antigen which is heterologous to theBoHV-1 genome inserted between two converging BoHV-1 genes, wherein theinsertion does not substantially down-regulate expression of the BoHV-1genes, wherein the genetic material encoding the at least one antigen isinserted between the polyadenylation signals of two converging genesselected from the group consisting of UL41 and UL40, UL36 and UL35, UL22and UL21, UL19 and UL15, UL11 and UL10 and UL8 and UL7.
 2. The vaccineof claim 1 wherein the site of insertion is between UL41 and UL40. 3.The vaccine of claim 1 wherein the functionally equivalent site isbetween UL11 and UL10.
 4. The vaccine of claim 1 wherein the siteinsertion is between UL8 and UL7.
 5. The vaccine of claim 1 wherein thegenetic material encoding the at least one antigen is inserted into theBoHV-1 genome via an inducible recombination system.
 6. The vaccine ofclaim 5 wherein the inducible recombination system is GET recombination.7. The vaccine of claim 1 wherein the BoHV-1 produces a BoHV-1 antigen.8. The vaccine of claim 1 wherein the at least one antigen is selectedfrom the group consisting of an antigen from bovine viral diarrhea virus(BVDV) and an antigen from a microorganism.
 9. The vaccine of claim 8wherein the BVDV antigen is selected from the group consisting ofglycoprotein E0 and glycoprotein E2.
 10. The vaccine of claim 8 whereinthe microorganism is selected from the group consisting of Mycoplasmabovis, a Salmonella species, Pateurella multocida, Manhiemia haemolyticaand Haemophilus somnus.
 11. The vaccine of claim 1, formulated for nasaladministration.
 12. A method of producing a vaccine against at least oneantigen from a bovine pathogen, said method comprising: (i)incorporating a BoHV-1 genome from a low virulence BoHV-1 into abacterial artificial chromosome (BAC) vector to form a BoHV-1 pre-vectorBAC construct; (ii) inserting genetic material encoding the at least oneantigen into the BoHV-1 pre-vector BAC construct via an induciblerecombination system to generate a recombinant BoHV-1-BAC (rBoHV-1-BAC)vector wherein the genetic material encoding the at least one antigen isinserted between the polyadenylation signals of two converging genes ata functionally equivalent site in another BoHV-1 to a site selected fromthe group consisting of between convergent genes UL41 and UL40, UL36 andUL35, UL22 and UL21, UL19 and UL15, UL11 and UL10 and UL8 and UL7; (iii)transforming and amplifying the rBoHV-1-BAC vector in a bacterial host;and (iv) purifying and isolating the rBoHV-1-BAC vector from thebacterial host and formulating the vector into a vaccine composition.13. The method of claim 12 wherein the site of insertion is between UL41and UL40.
 14. The method of claim 12 wherein the site of insertion isbetween UL11 and UL10.
 15. The method of claim 12 wherein the site ofinsertion is between UL8 and UL7.
 16. The method of claim 12 wherein theat least one antigen is selected from the group consisting of an antigenfrom bovine viral diarrhea virus (BVDV) and an antigen from amicroorganism.
 17. A cultured cell transfected with the rBoHV-1-BACvector of claim 12.